Regulation of sodium channels by plunc proteins

ABSTRACT

The present invention relates to the ability of PLUNC proteins, such as SPLUNC1 and SPLUNC2, to bind to sodium channels and inhibit activation of the sodium channels. The invention further relates to methods for regulating of sodium absorption and fluid volume and treating disorders responsive to modulating sodium absorption by modulating the binding of PLUNC proteins to sodium channels.

RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 13/321,617, now allowed, filed Jan. 26, 2012, whichis a 35 U.S.C. §371 national phase application of PCT ApplicationPCT/US2010/036531, filed May 28, 2010, which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No.61/182,287, filed May 29, 2000. The entire content of each of theseapplications is incorporated herein by reference.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled 5470-527TSCT_ST25.txt, 6,617 bytes in size, generatedon Dec. 1, 2016 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to the ability of PLUNC proteins, such asSPLUNC1 and SPLUNC2, to bind to sodium channels and inhibit activationof the sodium channels. The invention further relates to methods forregulating of sodium absorption and fluid volume and treating disordersresponsive to modulating sodium absorption by modulating the binding ofPLUNC proteins to sodium channels.

BACKGROUND OF THE INVENTION

Epithelial mucosal surfaces are lined with fluids whose volume andcomposition are precisely controlled. In the airways, a thin film ofairway surface liquid helps protect mammalian airways from infection byacting as a lubricant for efficient mucus clearance (Chmiel et al.,Respir. Res. 4:8 (2003); Knowles et al., J. Clin. Invest. 109:571(2002)). This layer moves cephalad during mucus clearance and excessliquid that accumulates as two airways converge is eliminated by Na⁺-ledairway surface liquid absorption with Na⁺ passing through the epithelialNa⁺ channel (ENaC) (Knowles et al., J. Clin. Invest. 109:571 (2002)).How ENaC activity is sensed and controlled by the airways is poorlyunderstood. However, there is evidence that reporter molecules in theairway surface liquid can serve as volume sensing signals whose dilutionor concentration can alter specific cell surface receptors which controlion transport rates to either absorb or secrete airway surface liquid asneeded (Chambers et al., Respir. Physiol. Neurobiol. 159:256 (2007).ENaC must be cleaved by intracellular furin-type proteases and/orextracellular channel activating proteases (CAPs) such as prostasin tobe active and to conduct Na⁺ (Planes et al., Curr. Top. Dev. Biol.778:23 (2007); Rossier, Proc. Am. Thorac. Soc. 1:4 (2004); Vallet etal., Nature 389:607 (1997); Chraibi et al., J. Gen. Physiol. 111:127(1998)). ENaC can also be cleaved and activated by exogenous serineproteases such as trypsin an action that is attenuated by the proteaseinhibitor aprotinin (Vallet et al., Nature 389:607 (1997)). When humanbronchial epithelial cultures are mounted in Ussing chambers wherenative airway surface liquid is washed away, ENaC is predominantlyactive, suggesting that cell attached proteases are predominant (Bridgeset al., Am. J. Physiol. Lung Cell. Mol. Physiol. 281:L16 (2001);Donaldson et al., J. Biol. Chem. 277:8338 (2002)). In contrast, underthin film conditions, where native airway surface liquid is present,ENaC activity is reduced, suggesting that airway surface liquid containssoluble proteases inhibitors (Myerburt et al., J. Biol Chem. 281:27942(2006); Tarran et al., J. Gen. Physiol. 127:591 (2006)).

The Palate Lung and Nasal epithelial Clone (PLUNC) family are secretedproteins that are subdivided into short (SPLUNCs) and long (LPLUNCs)members which contain either one or two domains respectively (Bingle etal., Biochim. Biophys. Acta 1493:363 (2000); Weston et al., J. BiolChem. 274:13698 (1999)). The original PLUNC gene which is now calledSPLUNC1 comprises up to 10% of total protein in the airway surface lipidand can readily be detected in both nasal lavage and tracheal secretions(Bingle, C. D., and Craven, C. J. (2002) PLUNC: a novel family ofcandidate host defense proteins expressed in the upper airways andnasopharynx Hum Mol Genet 11, 937; Campos, M. A., et al. (2004)Purification and characterization of PLUNC from human tracheobronchialsecretions Am J Respir Cell Mol Biol 30, 184; Lindahl M., Stahlbom, B.,and Tagesson, C. (2001) Identification of a new potential airwayirritation marker, palate lung nasal epithelial clone protein, in humannasal lavage fluid with two-dimensional electrophoresis andmatrix-assisted laser desorption/ionization-time of flightElectrophoresis 22, 1795). SPLUNC1 is expressed in both submucosalglands, the superficial epithelia and in neutrophils and in theory, ispresent in the correct regions of the lung to be a volume sensingmolecule since it can be secreted onto the mucosal surface of thesuperficial epithelial where ENaC is expressed (Bartlett et al., J.Leukoc. Biol. 83:1201 (2008); Bingle et al., J. Pathol. 205:491 (2005)).

The present inventien addresses previous shortcomings in the art bydisclosing the regulation of sodium channels by PLUNC proteins and themanipulation of this pathway to regulate sodium absorption and fluidvolume and treat disorders responsive to modulating sodium absorption.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of the abilityof PLUNC proteins to regulate the activity of sodium channels.Accordingly, in one aspect the invention relates to a method ofinhibiting the activation of a sodium channel, comprising contacting asodium channel with a PLUNC protein of a functional fragment thereofthereof. In one embodiment, the sodium channel is an epithelial sodiumchannel (ENaC). In another embodiment, the PLUNC protein is SPLUNC1 orSPLUNC2. In one embodiment, the PLUNC protein or a functional fragmentthereof binds to the sodium channel.

Another aspect of the invention relates to a method of inhibiting sodiumabsorption through a sodium channel, comprising contacting the sodiumchannel with a PLUNC protein or a functional fragment thereof. In oneembodiment, the PLUNC protein or a functional fragment thereof binds tothe sodium channel.

A further aspect of the invention relates to a method of increasing thevolume of fluid lining an epithelial mucosal surface, comprisingcontacting a sodium channel present on the epithelial mucosal surfacewith a PLUNC protein or a functional fragment thereof. In oneembodiment, the PLUNC protein or a functional fragment thereof binds tothe sodium channel.

Another aspect of the invention relates to a method of reducing thelevel of a sodium channel present on the surface of a cell, comprisingcontacting the sodium channel with a PLUNC protein or a functionalfragment thereof. In one embodiment, the PLUNC protein or a functionalfragment thereof binds to sodium channel.

A further aspect of the invention relates to a method of treating adisorder responsive to inhibition of sodium absorption across anepithelial mucosal surface in a subject in need thereof, comprisingdelivering to the subject a therapeutically effective amount of a PLUNCprotein or a functional fragment thereof. In one embodiment, the PLUNCprotein or a functional fragment thereof binds to the sodium channel.

Another aspect of the invention relates to a method of regulating saltbalance, blood volume, and/or blood pressure in a subject in needthereof, comprising delivering to the subject a therapeuticallyeffective amount of a PLUNC protein or a functional fragment thereof. Inone embodiment, the PLUNC protein or a functional fragment thereof bindsto the sodium channel.

An additional aspect of the invention relates to a method of increasingthe activation of a sodium channel, comprising inhibiting the binding ofa PLUNC protein to the sodium channel.

A further aspect of the invention relates to a method of increasingsodium absorption through a sodium channel, comprising inhibiting thebinding of a PLUNC protein to the sodium channel, thereby activating thesodium channel.

Another aspect of the invention relates to a method of decreasing thevolume of fluid lining an epithelial mucosal surface, comprisinginhibiting the binding of a PLUNC protein to a sodium channel present inthe epithelial mucosal surface, thereby activating the sodium channel.

A further aspect of the invention relates to a method of increasing thelevel of a sodium channel present on the surface of a cell, comprisinginhibiting the binding of a PLUNC protein to the sodium channel presenton the surface of the cell.

An additional aspect of invention relates to a method of treating adisorder responsive to activation of sodium absorption in a subject inneed thereof, comprising inhibiting the activity of a PLUNC protein inthe subject.

Another aspect of the invention relates to a method of regulating saltbalance, blood volume, and/or blood pressure in a subject in needthereof, comprising inhibiting the activity of a PLUNC protein in thesubject.

A further aspect of the invention relates to a method of enhancing thesense of taste in a subject, comprising inhibiting the activity of aPLUNC protein in the subject.

An additional aspect of the invention relates to polypeptide consistingessentially of the sodium channel binding domain of a PLUNC protein, aswell as a polynucleotide encoding the polypeptide and a vector and/orcell comprising the polynucleotide.

Another aspect of the invention relates to a compound that mimics thesodium channel binding domain of a PLUNC protein and binds to a sodiumchannel, wherein cleavage of the sodium channel by a protease isinhibited when bound to the compound.

A further aspect of the invention relates to a polypeptide consistingessentially of a PLUNC protein binding domain of a sodium channel, aswell as a polynucleotide encoding the polypeptide and a vector and/orcell comprising the polynucleotide.

An additional aspect of the invention relates to a compound that mimicsa PLUNC protein binding domain of a sodium channel and binds to a PLUNCprotein, wherein binding of PLUNC protein to the sodium channel isinhibited when bound to the compound.

Another aspect of the invention relates to a kit comprising thepolypeptide, polynucleotide, vector, cell peptidomimetic, or compound ofthe invention.

Another aspect of the invention relates to the use of a PLUNC protein ora functional fragment thereof for the preparation of a medicament totreat a disorder responsive to inhibition of sodium absorption in asubject in need thereof.

Another aspect of the invention relates to the use of an inhibitor of aPLUNC protein for the preparation of a medicament to treat a responsiveto activation of sodium absorption in a subject in need thereof.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that SPLUNC1 is present in the airway surface liquid ofhuman bronchial cultures. Airway surface liquid was incubated withtrypsin-agarose beads±aprotinin and proteins were separated on 15% SDSgel and visualized with a silver stain. The outlined bands were then cutout and analyzed by MALDI-MS/MS and the proteins identified are shown inTable 2. SPLUNC1 was detected in Bands 1 & 2, and its binding to trypsinwas attenuated in the presence of aprotinin.

FIGS. 2A-2E show the presence and function of SPLUNC1 in airway fluid.

FIG. 3 shows that SPLUNC1 is cleaved by trypsin. A 60 min treatment with1 U/ml trypsin caused both a ˜1 kDa and a 10 kDa shift in SPLUNC1 size.SPLUNC1 was labeled C-terminally with a V5 tag and detected with ananti-V5 antibody. SPLUNC1 cleavage products are shown with arrows.

FIG. 4 shows that SPLUNC1 affects the transepithelial resistance (Rt) ina similar fashion to amiloride. Human bronchial epithelial cultures werewashed 5× with PBS over 1 hr to remove any native SPLUNC1 and thenexposed to 50 ng/ml recombinant SPLUNC1 for 30 min or 100 mM amiloridefor 10 min or SPLUNC1 followed by amiloride. All n=12. *=p<0.05different to control.

FIGS. 5A-5C show the effect of expressing SPLUNC1 and ENaC in Xenopusoocytes.

FIGS. 6A-6B show the effect of SPLUNC1 on cleavage of ENaC.

FIGS. 7A-7E show the effect of expressing SPLUNC1 and ENaC is Xenopusoocytes.

FIG. 8 shows a Western blot showing αENaC expression in JME cells stablytransfected with pQCXIN-α-ENaC-YFP but not in cells from the samepassage infected with the empty pQCXIN vector

FIGS. 9A-9B show binding of SPLUNC1 to ENaC.

FIG. 10 shows that SPLUNC1 binds to α, β and γ ENaC subunits. Oocyteswere coinjected with 0.3 ng αβγ ENaC subunits±SPLUNC1 (1 ng). Gels showrepresentative co-immunoprecipitation of V5-tagged SPLUNC1 and HA-taggedENaC subunits. Arrowheads denote ENaC or SPLUNC1 bands and U.I. denotestransfected control oocytes.

FIGS. 11A-11E show the inhibition of SPLUNC1 with shRNA.

FIG. 12 shows that SPLUNC1 is highly expressed in the trachea, colon andkidney, was obtained from whole trachea, kidney, stomach and colon vs.specific SPLUNC1 cDNA.

FIG. 13 shows the effect of PLUNC family members on ENaC activity.

FIG. 14 shows that the ability of SPLUNC1 to inhibit the transepithelialPD is attenuated by DTT pretreatment in primary human bronchialepithelial cultures. Cultures were prewashed to remove endogenousSPLUNC1 and the basal PD was measured (control, ctrl), then either 50ng/ml recombinant SPLUNC1 or recombinant SPLUNC1 that had been reducedwith DTT was added, and the PD was remeasured on the same cultures 45min later, *=p<0.05 different from control. †=p<0.05 different toSPLUNC1 alone.

FIG. 15 shows a Western blot run under non-denaturing conditions showingthat reduce (i.e., DTT-treated) SPLUNC1 migrates along the gel at adifferent rate to non-denatured SPLUNC1.

FIGS. 16A-16B show that SPLUNC1 may inhibit ENaC by decreasing thenumber of ENaC channels in the plasma membrane. A, Surface biotinylationof αENaC shows that plasma membrane ENaC is decreased followingcoexpression with SPLUNC1 in oocytes. 1, control; 2, αENaC, 3, αENaC &SPLUNC1. Total lysate per lanes as 3-4 eggs run on a 10% Gel. B,addition of MTSET to ENaC containing the βS518C mutant increases ENaCP_(o) to 1.0 when coexpressed in oocytes yet the overall current isstill reduced by SPLUNC1 expression, suggesting that ENaC has beeninternalized. Open bars, control. Closed bars, MTSET addition. All n=6.

FIG. 17A shows the structure of SPLUNC1 and SPLUNC1 mutants examined forfunction and a comparison of the SPLUNC1 amino acid sequence (aminoacids 22-39 of SEQ ID NO:1) to the sequence of the α26 and γ43 subunitsof ENaC (SEQ ID NOS:9 and 10).

FIG. 17B shows a comparison of ENaC inhibition by full-length andtruncated SPLUNC1 at pH 7.4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in more detail withreference to the accompanying drawings, in which preferred embodimentsof the invention shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which this invention belongs. The terminology used in the descriptionof the indention herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.All publications, patent applications, patents, patent publications andother references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which reference is presented.

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ directions from left to right, unless specifically indicatedotherwise, nucleotides and amino acids are represented herein in themanner recommended by the IUPAC-IUB Biochemical Nomenclature Commission,or (for amino acids) by either the one-letter code, or the three lettercode, both in accordance with 37 C.F.R. §1.822 and established usage.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for cloning genes, amplifying and detectingnucleic acids, and the like. Such techniques are known to those skilledin the art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Ausubel et al. CurrentProtocols in Molecular Biology (Green Publishing Associates, Inc. andJohn Wiley & Sons, Inc., New York).

I. Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

Also as used herein, “and/or” refers, to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

The term “consists essentially of” (and grammatical variants), asapplied to a polynucleotide or polypeptide sequence of this invention,means a polynucleotide or polypeptide that consists of both the recitedsequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5′and/or 3′ or N-terminal and/or C-terminal ends of the recited sequencesuch that the function of the polynucleotide or polypeptide is notmaterially altered. The total of ten or less additional nucleotides oramino acids includes the total number of additional nucleotides or aminoacids on both ends added together. The term “materially altered,” asapplied to polynucleotides of the invention, refers to an increase ordecrease in ability to express the encoded polypeptide of at least about50% or more as compared to the expression level of a polynucleotideconsisting of the recited sequence. The term “materially altered,” asapplied to polypeptides of the invention, refers to an increase ordecrease in binding activity (e.g., to a sodium channel or PLUNCprotein) of at least about 50% or more as compared to the activity of apolypeptide consisting of the recited sequence.

The term “modulate,” “modulates,” or “modulation” refers to enhancement(e.g., an increase) or inhibition (e.g., a decrease) in the specifiedlevel or activity.

The term “enhance” or “increase” refers to an increase in the specifiedparameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen fold.

The term “inhibit” or “reduce” or grammatical variations thereof as usedherein refers to a decrease or diminishment in the specified level oractivity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%,95% or more. In particular embodiments, the inhibition or reductionresults in little or essentially no detectible activity (at most, aninsignificant amount, e.g., less than bout 10% or even 5%).

The term “contact” or grammatical variations thereof as used withrespect to a PLUNC protein and a sodium channel, refers to bringing thePLUNC protein and the sodium channel in sufficiently close proximity toeach other for one to exert a biological effect on the other. In someembodiments, the term contact means binding of the PLUNC protein to thesodium channel.

A “therapeutically effective” amount as used herein is as amount thatprovides some improvement or benefit to the subject. Alternativelystated, a “therapeutically effective” amount is an amount that willprovide some alleviation, mitigation, or decrease in at least oneclinical symptom in the subject. Those skilled, in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

By the terms “treat,” “treating,” or “treatment of,” it is intended thatthe severity of the subject's condition is reduced or at least partiallyimproved or modified and that some alleviation, mitigation or decreasein at least one clinical symptom is achieved.

As used herein, “nucleic acid,” “nucleotide sequence,” and“polynucleotide” are used interchangeably and encompass both RNA andDNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemicallysynthesized) DNA or RNA and chimeras of RNA and DNA. The term nucleicacid refers to a chain of nucleotides without regard to length of thechain. The nucleic acid can be double-stranded or single-stranded. Wheresingle-stranded, the nucleic acid can be a sense strand or an antisensestrand. The nucleic acid can be synthesized using oligonucleotideanalogs or derivatives (e.g., inosine or phosphorothioate nucleotides).Such oligonucleotides can be used, for example, to prepare nucleic acidsthat have altered base-pairing abilities or increased resistance tonucleases. The present invention further provides a nucleic acid that isthe complement (which can be either a full complement or a partialcomplement) of a nucleic acid or nucleotide sequence of this invention.

An “isolated polynucleotide” is a nucleotide sequence (e.g., DNA or RNA)that is not immediately contiguous with nucleotide sequences with whichit is immediately contiguous (one on the 5′ end and one on the 3′ end)in the naturally occurring genome of the organism from which it isderived. Thus, in one embodiment, an isolated nucleic acid includes someor all of the 5′ non-coding (e.g., promoter) sequences that areimmediately contiguous to a coding sequence. The term thereforeincludes, for example, a recombinant DNA that is incorporated into avector, into an autonomously replicating plasmid or virus, or into thegenomic DNA of a prokaryote or eukaryote, or which exists as a separatemolecule (e.g., a cDNA or a genomic DNA fragment produced by PCR orrestriction endonuclease treatment), independent of other sequences. Italso includes a recombinant DMA that is part of a hybrid nucleic acidencoding an additional polypeptide or peptide sequence. An isolatedpolynucleotide that includes a gene is not a fragment of a chromosomethat includes such gene, but rather includes the coding region andregulatory regions associated with the gene, but no additional genesnaturally found on the chromosome.

The term “isolated” can refer to a nucleic acid, nucleotide sequence orpolypeptide that is substantially free of cellular material, viralmaterial, and/or culture medium (when produced by recombinant DNAtechniques), or chemical precursors or other chemicals (when chemicallysynthesized). Moreover, an “isolated fragment” is a fragment of anucleic acid, nucleotide sequence or polypeptide that is not naturallyoccurring as a fragment and would not be found in the natural state.“Isolated” does not mean that the preparation is technically pure(homogeneous), but it sufficiently pure to provide the polypeptide ornucleic acid in a form in which it can be used for the intended purpose.

An isolated cell refers to a cell that is separated from othercomponents with which it is normally associated in its natural state.For example, an isolated cell can be a cell in culture medium and/or acell in a pharmaceutically acceptable carrier of this invention. Thus,an isolated cell can be delivered to and/or introduced into a subject.In some embodiments an isolated cell can be a cell that is removed froma subject and manipulated as described herein ex vivo and then returnedto the subject.

The term “fragment,” as applied to a polynucleotide, will be understoodto mean a nucleotide sequence of reduced length relative to a referencenucleic acid or nucleotide sequence and comprising, consistingessentially of, and/or consisting of a nucleotide sequence of contiguousnucleotides identical or almost identical, (e.g., 90%, 92%, 95%, 98%,99% identical) to the reference nucleic acid or nucleotide sequence.Such a nucleic acid fragment according to the invention may be, whereappropriate, included in a larger polynucleotide of which is aconstituent. In some embodiments, such fragments can comprise, consistessentially of, and/or consist of oligonucleotides having a length of atleast about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, or more consecutive nucleotides of a nucleic acid or nucleotidesequence according to the invention. In other embodiments, suchfragments can comprise, consist essentially of, and/or consist ofoligonucleotides having a length of less than about 200, 150, 100, 75,60, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, or less consecutivenucleotides of a nucleic acid or nucleotide sequence according to theinvention.

The term “fragment,” as applied to a polypeptide, will be understood tomean an amino acid sequence of reduced length relative to a referencepolypeptide or amino acid sequence and comprising, consistingessentially of, and/or consisting of an amino acid sequence ofcontiguous amino acids identical or almost identical (e.g., 90%, 92%,95%, 98%, 99% identical) to the reference polypeptide of amino acidsequence. Such a polypeptide fragment according to the invention may be,where appropriate, included in a larger polypeptide of which it is aconstituent. In some embodiments, such fragments can comprise, consistessentially of and/or consist of peptides having a length of at least:about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, or more consecutive amino acids of a polypeptide or amino acidsequence according to the invention. In other embodiments, suchfragments can comprise, consist essentially of and/or consist ofpeptides having a length of less than about 200, 150, 100, 75, 60, 50,45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 6, 4, or less consecutive aminoacids of a polypeptide or amino acid sequence according to theinvention.

A “vector” is any nucleic acid molecule for the cloning of and/ortransfer of a nucleic acid into a cell. A vector may be a replicon towhich another nucleotide sequence may be attached to allow forreplication of the attached nucleotide sequence. A “replicon” can be anygenetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome)that functions as an autonomous unit of nucleic acid replication invivo, i.e., capable of replication under its own control. The term“vector” includes both viral and nonviral (e.g., plasmid) nucleic acidmolecules introducing a nucleic acid into a cell in vitro, ex vivo,and/or in vivo. A large number of vectors known in the art may be usedto manipulate nucleic acids, incorporate response elements and promotersinto genes, etc. For example, the insertion of the nucleic acidfragments corresponding to response elements and promoters into asuitable vector can be accomplished by ligating the appropriate nucleicacid fragments into a chosen vector that has complementary cohesivetermini. Alternatively, the ends of the nucleic acid molecules may beenzymatically modified or any site may be produced by ligatingnucleotide sequences (linkers) to the nucleic acid termini. Such vectorsmay he engineered to contain sequences encoding selectable markers thatprovide the selection of cells that contain the vector and/or haveincorporated the nucleic acid of the vector into the cellular genome.Such markers allow identification and/or selection of host cells thatincorporate and express the proteins encoded by the market. A“recombinant” vector refers to a viral or non-viral vector thatcomprises one or more heterologous nucleotide sequences (i.e.,transgenes), e.g., two, three, four, five or more heterologousnucleotide sequences.

Viral vectors have been used in a wide variety of gene deliveryapplications in cells, as well as living animal subjects. Viral vectorsthat can be used include, but are not limited to, retrovirus,lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus,vaccinia virus, herpes virus, Epstein-Barr virus, and adenovirusvectors. Non-viral vectors include plasmids, liposomes, electricallycharged lipids (cytofectins), nucleic acid-protein complexes, andbiopolymers. In addition to a nucleic acid of interest, a vector mayalso comprise one or more regulatory regions, and/or selectable markersuseful in selecting, measuring, and monitoring nucleic acid transferresults (delivery to specific tissues, duration of expression, etc.).

Vectors may be introduced into the desired cells by methods known in theart, e.g., transfection, electroporation, microinjection, transduction,cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a nucleic acid vectortransporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu etal., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., CanadianPatent Application No. 2,012,311, filed Mar. 15, 1090).

In some embodiments, a polynucleotide of this invention can be deliveredto a cell in vivo by lipofection. Synthetic cationic lipids designed tolimit the difficulties and dangers encountered with liposome-mediatedtransfection can be used to prepare liposomes for in vivo transfectionnucleotide sequence of this invention (Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413 (1987); Mackey, et al., Proc. Natl. Acad. Sci. U.S.A.85:8027 (1988); and Ulmer et al., Science 259:1745 (1993)). The use ofcationic lipids may promote encapsulation of negatively charged nucleicacids, and also promote fusion with negatively charged cell membranes(Felgner et al., Science 337:387 (1989)). Particularly useful lipidcompounds and compositions for transfer of nucleic acids are describedin International Patent Publications WO95/18863 and WO96/17823, and inU.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenousnucleotide sequences into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly preferred in a tissuewith cellular heterogeneity, such as pancreas, liver, kidney, and thebrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting (Mackey, et al., 1988, supra). Targeted peptides,e.g., hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules can be coupled to liposomes chemically.

In various embodiments, other molecules can be used for facilitatingdelivery of a nucleic acid in vivo, such as a cationic oligopeptide(e.g., WO95/21931), peptides derived from nucleic acid binding proteins(e.g., WO96/25508), and/or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as naked nucleic acid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated nucleic acid delivery approaches can also be used(Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem.262:4429 (1987)).

The term “transfection” or “transduction” means the uptake of exogenousor heterologous nucleic acid (RNA and/or DNA by a cell. A cell has been“transfected” or “transduced” with an exogenous or heterologous nucleicacid when such nucleic acid has been introduced or delivered inside thecell. A cell has been “transformed” by exogenous or heterologous nucleicacid when the transfected or transduced nucleic acid imparts aphenotypic change in the cell and/or a change in an activity or functionof the cell. The transforming nucleic acid can integrated (covalentlylinked) into chromosomal DNA making up the genome of the cell or it canbe present as a stable plasmid.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and encompass both peptides and proteins, unlessindicated otherwise.

A “fusion protein” is a polypeptide produced when two heterologousnucleotide sequences or fragments thereof coding for two (or more)different polypeptides not found fused together in nature are fusedtogether in the correct translational reading frame. Illustrative fusionpolypeptides include fusions of a polypeptide of the invention (or afragment thereof) to all or a portion of glutathione-S-transferase,maltose-binding protein, or a reporter protein (e.g., Green FluorescentProtein, β-glucuronidase, β-galactosidase, luciferase, etc.),hemagglutinin, c-myc, FLAG epitope, etc.

As used herein, a “functional” polypeptide of “functional fragment” isone that substantially retains at least one biological activity normallyassociated with that polypeptide binding to or inhibiting a sodiumchannel or a PLUNC protein). In particular embodiments, the “functional”polypeptide or “functional fragment” substantially retains ail of theactivities possessed by the unmodified peptide. By “substantiallyretains” biological activity, it is meant that the polypeptide retainsat least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%,99%, or more, of the biological activity of the native polypeptide (andcan even have a higher level of activity than the native polypeptide). A“non-functional” polypeptide is one that exhibits little or essentiallyno detectable biological activity normally associated with thepolypeptide (e.g., at most, only an insignificant amount, e.g., lessthan about 10% or even 5%). Biological activities such as proteinbinding and angiogenic activity can be measured using assays that arewell known in the art and as described herein. In some embodiments, theterm “functional fragment” also encompasses compound that are mimeticsof a portion of the polypeptide (e.g., peptidomimetics) that have atleast one biological activity that is substantially the same as anactivity associated with the polypeptide.

By the term “express” or “expression” of a polynucleotide codingsequence, it is meant that the sequence is transcribed, and optionally,translated. Typically, according to the present invention, expression ofa coding sequence of the invention will result in production of thepolypeptide of the invention. The entire expressed polypeptide orfragment can also function in intact cells without purification.

The term “about,” as used herein when referring to a measurable valuesuch as an amount of polypeptide, dose, time, temperature, enzymaticactivity or other biological activity and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of thespecific amount.

II. Decreasing Sodium Channel Activation

A first aspect of the invention relates to the ability of PLUNC proteinsto bind to a sodium channel and prevent activation of the sodiumchannel, thereby inhibiting the flow of sodium ions. Thus, one aspect ofthe present invention relates to a method of inhibiting the activationof a sodium channel, comprising contacting (e.g., binding) a sodiumchannel with a PLUNC protein or a functional fragment thereof. In oneembodiment, the sodium channel is an epithelial sodium channel (ENaC),e.g., human ENaC. In another embodiment, the sodium channel is one thatis similar in sequence and/or structure to ENaC, such as acid-sensingion channels (ASIC). The inhibition of sodium channel activation can bemeasured by any method known in the art or disclosed herein, including,without limitation, measuring sodium flow or change in potential acrossa membrane, across a cell, or across a natural or artificial lining. Theinhibition can be least about 20%, e.g., at least about 30%, 40%, 50%,660%, 70%, 80%, 90%, or 100%.

The PLUNC protein can be any protein from the PLUNC family that can bindto and inhibit the activation of sodium channel. In one embodiment, thePLUNC protein is a human PLUNC protein. In another embodiment, the PLUNCprotein is SPLUNC1 or SPLUNC2.

The method of inhibiting the activation of a sodium channel can becarried out, e.g., on an isolated sodium channel, a sodium channel in anartificial membrane, or a sodium channel in a cell. In one embodiment,the sodium channel is present in an isolated cell, e.g., a culturedprimary cell or cell line. In another embodiment, the isolated cell ispart of an epithelial cell culture, e.g., a natural or artificialepithelial lining, e.g., a cell culture in a device (such as an Ussingchamber) in which characteristics such as ion flow and/or potential canbe measured across lining. In another embodiment, the cell is part of anisolated tissue or a tissue culture. In a further embodiment, the cellcan be present in an animal, e.g., an animal that is a disease model ora subject in need of treatment.

In one embodiment, the step of contacting (e.g., binding) the sodiumchannel with a PLUNC protein comprises delivering the PLUNC protein of afunctional fragment or homolog thereof to a cell comprising the sodiumchannel. In another embodiment, the contacting step (e.g., binding)comprises delivering a polynucleotide encoding the PLUNC protein or afunctional fragment or homolog thereof to a cell comprising the sodiumchannel.

As used herein, the term “homolog” is used to refer to a polypeptidewhich differs from a naturally occurring polypeptide by minormodifications to the naturally occurring polypeptide, but whichsignificantly retains a biological activity of the naturally occurringpolypeptide. Minor modifications include, without limitation, changes inone or a few amino acid side chains, changes: to one or a few aminoacids (including deletions, insertions, and substitutions), changes instereochemistry of one or a few atoms, and minor derivatizations,including, without limitation, methylation, glycosylation,phosphorylation, acetylation, myristoylation, prenylation, palmitation,amidation, and addition of glycosylphosphatidyl inositol. The term“substantially retains,” used herein, refers to a fragment, homolog, orother variant of a polypeptide that retains at least about 20% of theactivity of the naturally occurring polypeptide binding to a sodiumchannel), e.g., about 30%, 40%, 50% or more. Other biologicalactivities, depending on the polypeptide, may include enzyme activity,receptor binding, ligand binding, induction of a growth factor, a cellsignal transduction event, etc.

In one embodiment, the method comprises delivering to a cell comprisinga sodium channel an isolated PLUNC protein. In exemplary embodiments,the PLUNC protein comprises, consists essentially of, or consists of thepublicly known amino acid sequence of the PLUNC protein (e.g., asdisclosed in GenBank and disclosed herein) or a functional fragmentthereof. In another embodiment, the isolated PLUNC protein comprises,consists essentially of, or consists of an amino acid sequence that isat least 70% identical, e.g., at least 75%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the publicly known amino acid sequence or afunctional fragment thereof.

The amino acid sequence of human SPLUNC1 (SEQ ID NO:1) and human SPLUNC2(SEQ ID NO:2) are disclosed below. The conserved cysteine residues thatare spaced 43 amino acids apart and may be important for activity ateindicated.

SPLUNC1        10         20         30         40         50         60MFQTGGLIVF YGLLAQTMAQ FGGLPVPLDQ TLPLNVNPAL PLSPTGLAGS LTNALSNGLL        70         80         90        100        110        120SGGLLGILEN LPLLDILKPG GGTSGGLLGG LLGKVTSVIP GLNNIIDIKV TDPQLLELGL       130        140        150        160        170        180VQSPDGHFLY VTIPLGIKLQ VNTPLVGASL LRLAVKLDIT AEILAVRDKQ ERIHLVLGDC       190        200        210        220        230        240THSPGSLQIS LLDGLGPLPI QGLLDSLTGT LNKVLPELVQ GNVCPLVNEV LRGLDITLVH       250  DIVNMLIHGL QFVIKV SPLUNC2        10         20         30         40         50         60MLQLWKLVLL CGVLTGTSES LLDNLGNDLS NVVDKLEPVL HEGLETVDNT LKGILEKLKV        70         80         90        100        110        120DLGVLQKSSA WQLAKQKAQE AEKLLNNVIS KLLPTNTDIF GLKISNSLIL DVKAEPIDDG       130        140        150        160        170        180KGLNLSFPVT ANVTVAQPII GQIINLKASL DLLTAVTIET DPQTHQPVAV LGECASDPTS       190        200        210        220        230        240ISLSLLDKHS QIINKFVNSV INTLKSTVSS LLQKEICFLI RIFIHSLDVN VIQQVVDNPQHKTQLQTLI

The PLUNC proteins of the invention also include functional portions orfragments. The length of the fragment is not critical as long as itsubstantially retains the biological activity of the polypeptide (e.g.,sodium channel binding activity). Illustrative fragments comprise atleast about 4, 6, 8, 10, 12, 15, 20, 25, 30,35, 40, 45, 50, 75, 100,150, 200, of more contiguous amino acids of a PLUNC protein. In otherembodiments, the fragment comprises no more than about 200, 150, 100,75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 6, or 4 contiguous aminoacids of a PLUNC protein. In one embodiment, the fragment comprises,consists essentially of, or consists of a sequence from about residue 20to about residue 41 of human SPLUNC1, e.g., about residue 22 to aboutresidue 39, or the corresponding sequence (e.g., the approximately 20amino acids immediately after the signal peptide) from another PLUNCprotein.

Likewise, those skilled its the art will appreciate that the presentinvention also encompasses fusion polypeptides (and polynucleotidesequences encoding the same) comprising a PLUNC protein (or a functionalfragment thereof). For example, it may be useful to express thepolypeptide (or functional fragment) as a fusion protein that can berecognizes by a commercially available antibody (e.g., FLAG motifs) oras a fusion protein that can otherwise be more easily purified (e.g., byaddition of a poly-His tail). Additionally, fusion proteins that enhancethe stability of the polypeptide may be produced, e.g., fusion proteinscomprising maltose binding protein (MBP) or glutathione-S-transferase.As another alternative, the fusion protein can comprise a reportermolecule. In other embodiments, the fusion protein can comprise apolypeptide that provides a function or activity that is the same as ordifferent from the activity of the polypeptide, e.g., a targeting,binding, or enzymatic activity or function.

Likewise, it will be understood that the polypeptides specificallydisclosed herein will typically tolerate substitutions in the amino acidsequence and substantially retain biological activity. To identifypolypeptides of the invention other than those specifically disclosedherein, amino acid substitutions may be based on any characteristicknown in the art, including the relative similarity or differences ofthe amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge size, and the like.

Amino acid substitutions other than those disclosed herein may beachieved by changing the codons of the DNA sequence (of RNA sequence),according to the following codon table:

TABLE 1 Amino Acid Codons Alanine Ala A GCA GCC GCG GCT Cysteine Cys CTGC TGT Aspartic acid Asp D GAC GAT Glutamic acid Glu E GAA GAGPhenylalanine Phe F TTC TTT Glycine Gly G GGA GGC GGG GGT Histidine HisH CAC CAT Isoleucine Ile I ATA ATC ATT Lysine Lys K AAA AAG Leucine LeuL TTA TTG CTA CTC CTG CTT Methionine Met M ATG Asparagine Asn N AAC AATProline Pro P CCA CCC CCG CCT Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGT Serine Ser S AGC ACT TCA TCC TCG TCT ThreonineThr T ACA ACC ACG ACT Valine Val V GTA GTC GTG GTT Tryptophan Trp W TGGTyrosine Tyr Y TAC TAT

In identifying amino acid sequences encoding polypeptides other thanthose specifically disclosed herein, the hydropathic index of aminoacids may be considered. The importance of the hydropathic amino acidindex in conferring interactive biologic function on a protein isgenerally understood in the art (see, Kyte and Doolittle, J. Mol. Biol.157:105 (1982), incorporating herein by reference in its entirety). Itis accepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle, id.),these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cystein/cystine (+2.5); methionine (+1.9); alanine(+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan(−0.9); tryosine (−1.3); proline (−1.6); histine (−3.2); glutamate(−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine(−3.9); and arginine (−4.5).

Accordingly, the hydropathic index of the amino acid (or amino acidsequence) may be considered when modifying the polypeptides specificallydisclosed herein.

It is also understood in the art that the substitution of amino acidscan be made on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(incorporated herein by reference in its entirety) states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (±3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Thus, the hydrophilicity of the amino acid (or amino acid sequence) maybe considered when identifying additional polypeptides beyond thosespecifically disclosed herein.

In embodiments of the invention, the polynucleotide encoding the PLUNCprotein (or functional fragment) will hybridize to the nucleic acidsequences encoding PLUNC proteins that are known in the art or fragmentsthereof under standard conditions as known by those skilled in the artand encode a functional polypeptide or functional fragment thereof.

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions (e.g., conditions represented by a wash stringency of 35-40%formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.;conditions represented by a wash stringency of 40-45% formamide with5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and conditionsrepresented by a wash stringency of 50% formamide with 5×Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42°° C., respectively) to thepolynucleotide sequences encoding the PLUNC protein or functionalfragments thereof specifically disclosed herein. See, e.g., Sambrook etal., A Molecular Cloning: A Laboratory Manual 2nd Ed, (Cold SpringHarbor, N.Y., 1989).

In other embodiments, polynucleotide sequences encoding the PLUNCprotein have at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or higher sequence identity with the publicly known nucleic acidsequences (disclosed in GenBank) or functional fragments thereof andencode a functional polypeptide or functional fragment thereof.

Further, it will be appreciated by those skilled in the art that therecan be variability in the polynucleotides that encode the polypeptides(and fragments thereof) of the present invention due to the degeneracyof the genetic code. The degeneracy of the genetic code, which allowsdifferent nucleic acid sequences to code for the same polypeptide, iswell known in the literature (See, e.g., Table 1).

Likewise, the polypeptides (and fragments thereof) of the inventioninclude polypeptides that have at least about 70%, 80%, 85%, 95%, 96%,97%, 98%, 99% or higher amino acid sequence identity with the publiclyknown polypeptide sequences.

As is known in the art, a number of different programs can be used toidentify whether a polynucleotide or polypeptide has sequence identityor similarity to a known sequence. Sequence identity or similarity maybe determined using standard techniques known in the art, including, butnot limited to, the local sequence identify algorithm of Smith &Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identityalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Natl.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program, described by Devereux et al.,Nucl. Acid Res. 12:387 (1984), preferably using the default settings, orby inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351 (1987); the method is similar to that described by Higgins &Sharp, CABIOS 5:151 (1089).

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215:403 (1990) and Karlin et al.,Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., Meth. Enzymol., 266:460(1996). blast.wustl/edu/blast/README.html.WU-BLAST-2 uses several search parameters, which are preferably set tothe default values. The parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched; however, the valuesmay be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., Nucleic Acids Res. 25:3389 (1997).

A percentage amino acid sequence identity value is determined by thenumber of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

In a similar manner, percent nucleic acid sequence identity with respectto the coding sequence of the polypeptides disclosed herein is definedas the percentage of nucleotide residues in the candidate sequence thatare identical with the nucleotides in the polynucleotide specificallydisclosed herein.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the polypeptides specifically disclosed herein.It is understood that in one embodiment the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than a sequence specificallydisclosed herein, will be determined using the number of amino acids inthe shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

Those skilled in the art will appreciate that the isolatedpolynucleotides encoding the polypeptides of the invention willtypically be associated with appropriate expression control sequences,e.g., transcription/translation control signals and polyadenylationsignals.

It will further be appreciated that a variety of promoter/enhancerelements can be used depending on the level and tissue-specificexpression desired. The promoter can be constitutive or inducible,depending on the pattern of expression desired. The promoter can benative or foreign and can be a natural or a synthetic sequence. Byforeign, it is intended that the transcriptional initiation region isnot found in the wild-type host into which the transcriptionalinitiation region is introduced. The promoter is chosen so that it willfunction in the target cell(s) of interest.

To illustrate, the polypeptide coding sequence can be operativelyassociated with a cytomegalovirus (CMV) major immediate-early promoter,an albumin promoter, an Elongation Factor 1-α (EF1-α) promoter, a PγKpromoter, a MFG promoter, or a Rous sarcoma virus promoter.

Inducible promoter/enhancer elements include hormone-inducible andmetal-inducible elements, and other promoters regulated by exogenouslysupplied compounds, including without limitation, the zinc-induciblemetallothionein (MT) promoter; the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter; the T7 polymerase promoter system(see WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl.Acad. Sci. USA 93:3346 (1996)); the tetracycline-repressible system(Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547 (1992)); thetetracycline-inducible system (Gossen et al., Science 268:1766 (1995);see also Harvey et al., Curr. Opin. Chem. Biol. 2:512 (1998)); theRU486-inducible system (Wang et al., Nat. Biotech. 15:239 (1997); Wanget al., Gene Ther., 4:432 (1997)); and the rapamycin-inducible system(Magari et al., J. Clin. Invest. 100:2865 (1997)).

Moreover, specific initiation signals, are generally required forefficient translation of inserted polypeptide coding sequences. Thesetranslational control sequences, which can include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic.

The present invention further provides cells comprising the isolatedpolynucleotides and polypeptides of the invention. The cell may becultured cell or a cell in vivo, e.g., for use in therapeutic methods,diagnostic methods, screening methods, methods for studying thebiological action of the PLUNC proteins, in methods of producing thepolypeptides, or in methods of maintaining or amplifying thepolynucleotides of the invention, etc. In another embodiment, the cellis an ex vivo cell that has been isolated from a subject. The ex vivomay be modified and then reintroduced into the subject for diagnostic ortherapeutic purposes.

In particular embodiments, the cell is an untransformed epithelial cellor a cell from an epithelial cell line.

The isolated polynucleotide can be incorporated into an expressionvector. Expression vectors compatible with various host cells are wellknown in the art and contain suitable elements for transcription andtranslation of nucleic acids. Typically, an expression vector containsan “expression cassette,” which includes, in the 5′ to 3′ direction, apromoter, a coding sequence encoding a PLUNC protein or sodium channelor functional fragment thereof operatively associated with the promoter,and, optionally, a termination sequence including a stop signal for RNApolymerase and a polyadenylation signal for polyadenylase.

Non-limiting examples of promoters of this invention include CYCI, HI83,GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI, and alkaline phosphatase promoters (useful for expression inSaccharomyces); AOX1 promoter (useful for expression in Pichia);β-lactamase, lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trcpromoters (useful for expression in Escherichia coli); light regulated-,seed specific-, pollen specific-, ovary specific-, pathogenesis ordisease related-promoters, cauliflower mosaic virus 35S, CMV 35Sminimal, cassaya vein mosaic virus (CsVMV), chlorophyll a/b bindingprotein, ribulose 1,5-bisphosphate carboxylase, shoot-specificpromoters, root specific promoters, chitinase, stress induciblepromoters, rice tungro bacilliform virus, plant super-promoter, potatoleucine aminopeptidase, nitrate reductase, mannopine synthase, nopalinesynthase ubiquitin, zein protein, and anthocyanin promoters (useful forexpression in plant cells).

Further examples of animal and mammalian promoters known in the artinclude, but are not limited to, the SV40 early (SV40e) promoter region,the promoter contained in the 3′ long terminal repeat (LTR) of Roussarcoma virus (RSV), the promoters of the EIA or major late promoter(MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) earlypromoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter,baculovirus IE1 promoter, elongation factor 1 alpha (EF1) promoter,phosphogylcerate kinase (PGK) promoter, ubiquitin (Ubc) promoter, analbumin promoter, the regulatory sequences of the mousemetallothionein-L promoter and transcriptional control regions, theubiquitous promoters (HPRT, vitmentin, α-actin, tubulin and the like),the promoters of the intermediate filaments (desmin, neurofilaments,keratin, GFAP, and the like), the promoters of therapeutic genes (of theMDR, CFTR or factor VIII type, and the like), pathogenesis and/ordisease-related promoters, and promoters that exhibit tissuespecificity, such as the elastase I gene control region, which is activein pancreatic acinar cells; the insulin gene control region active inpancreatic beta cells, the immunoglobulin gene control region active inlymphoid cells, the mouse mammary tumor virus control region active intesticular, breast, lymphoid and mast cells; the albumin gene promoter,the Apo AI and Apo AII control regions active in liver, thealpha-fetoprotein gene control region active in liver, the alphaI-antitrypsin gene control region active in the liver, the beta-globingene control region active in myeloid cells, the myelin basic proteingene control region active in oligodendrocyte cells in the brain, themyosin light chain-2 gene control region active in skeletal muscle, andthe gonadotropic releasing hormone gene control region active in thehypothalamus; the pyruvate kinase promoter, the villin promoter, thepromoter of the fatty acid binding intestinal protein, the promoter ofsmooth muscle cell α-actin, and the like. In addition, any of theseexpression sequences of this invention can be modified by addition ofenhancer and/or regulatory sequences and the like.

Enhancers that may be used in embodiments of the invention include butare not limited to an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor I (EFI) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may be derived from various genes native to the preferredhosts. In some embodiments of the invention, the termination controlregion, may comprise or be derived from a synthetic sequence, asynthetic polyadenylation signal, an SV40 late polyadenylation signal anSV40 polyadenylation signal, a bovine growth hormone (BGH)polyadenylation signal, viral terminator sequences or the like.

It will be apparent to those skilled in the art that any suitable vectorcan be used to deliver the polynucleotide to a cell or subject. Thesector can be delivered to cells in vivo. In other embodiments, thevector can be delivered to cells ex vivo, and then cells containing thevector are delivered to the subject. The choice of delivery vector canbe made based on a number of factors known in the art, including age andspecies of the target host, in vitro versus in vivo delivery, level andpersistence of expression desired, intended purpose (e.g., for therapyor screening), the target cell or organ, route of delivery, size of theisolated polynucleotide, safety concerns, and the like.

Suitable vectors include plasmid vectors, viral vectors (e.g.,retrovirus, alphavirus; vaccinia virus; adenovirus, adeno-associatedvirus and other parvoviruses, lentivirus poxvirus, or herpes simplexvirus), lipid vectors, poly-lysine vectors, synthetic polyamino polymervectors, and the like.

Any viral vector that is known in the art can be used in the presentinvention. Protocols for producing recombinant viral vectors and forusing viral vectors for nucleic acid delivery can be found in Ausubel etal., Current Protocols in Molecular Biology (Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York) and otherstandard laboratory manuals (e.g., Vectors for Gene Therapy. In: CurrentProtocols in Human Genetics, John Wiley and Sons, Inc.; 1997).

Non-viral transfer methods can also be employed. Many non-viral methodsof nucleic acid transfer rely on normal mechanisms used by mammaliancells for the uptake and intracellular transport of macromolecules. Inparticular embodiments, non-viral nucleic acid delivery systems rely onendocytic pathways for the uptake of the nucleic acid molecule by thetargeted cell. Exemplary nucleic acid delivery systems of this typeinclude liposomal derived systems, poly-lysine conjugates, andartificial viral envelopes.

In particular embodiments, plasmid vectors are used in the practice ofthe present invention. For example, naked plasmids can be introducedinto muscle cells by injection into the tissue. Expression can extendover many months, although the number of positive cells is typically low(Wolff et al., Science 247:247 (1989)). Cationic lipids have beendemonstrated to aid in introduction of nucleic acids into some cells inculture (Felgner and Ringold, Nature 337:387 (1989)), Injection ofcationic lipid plasmid DNA complexes into the circulation of mice havebeen shown to result in expression of the DNA in lung (Brigham et al.,Am. J. Med. Sci. 298:278 (1989)). One advantage of plasmid DNA is thatit can be introduced into non-replicating cells.

In a representative embodiment, a nucleic acid molecule (e.g., aplasmid) can be entrapped in a lipid particle bearing positive chargeson its surface and, optionally, tagged with antibodies against cellsurface antigens of the target tissue (Mizuno et al., No Shinkei Geka20:547 (1992); PCT publication WO 91/06309; Japanese patent application1047381; and European patent publication EP-A-40375).

Liposomes that consist of amphiliphilic cationic molecules are useful asnon-viral vectors for nucleic acid delivery in vitro and in vivo(reviewed in Crystal, Science 270:404 (1995); Blaese et al., Cancer GeneTher. 2:291 (1995); Behr et al., Bioconjugate Chem. 5:382 (1994); Remyet al., Bioconjugate Chem. 5:647 (1994); and Gao et al., Gene Therapy2:170 (1995)). The positively charged liposomes are believed to complexwith negatively charged nucleic acids via electrostatic interactions toform lipid:nucleic acid complexes. The lipid:nucleic acid complexes haveseveral advantages as nucleic acid transfer vectors. Unlike viralvectors, the lipid:nucleic acid complexes can be used to transferexpression cassettes of essentially unlimited size. Since the complexeslack proteins, they can evoke fewer immunogenic and inflammatoryresponses. Moreover, they cannot replicate or recombine to form aninfectious agent and have low integration frequency. A number ofpublications have demonstrated that amphiphilic cationic lipids canmediate nucleic acid delivery in vivo and in vitro (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413 (1987); Loeffler et al., Meth.Enzymol. 217:599 (1993); Felgner et al., J. Biol. Chem. 269:2550(1994)).

Several groups have reported the use of amphiphilic cationic lipidnucleic acid complexes for in vivo transfection both in animals and inhumans (reviewed in Gao et al., Gene Therapy 2:710 (1995); Zhu et al.,Science 261:209 (1993); and Thierry et al., Proc. Natl. Acad. Sci. USA92:9742 (1995)). U.S. Pat. No. 6,410,049 describes a method of preparingcationic lipid:nucleic acid complexes that have a prolonged shelf life.

Expression vectors can be designed for expression of polypeptides inprokaryotic or eukaryotic cells. For example, polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (e.g.,. thebaculovirus expression system), yeast cells, plant cells or mammaliancells. Some suitable host cells are discussed further in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Examples of bacterial vectors include pQE70,pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK,pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3,pKK233-3, pDR540, and pRIT5 (Pharmacia). Examples of vectors forexpression in the yeast S. cerevisiae include pYepSecl (Baldari et al.,EMBO J. 6:229 (1987)), pMFa (Kurjan and Kerskowitz, Cell 30:933 (1992)),pJRY88 (Schultz et al., Gene 54:113 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.). Baculovirus vectors available forexpression of nucleic acids to produce proteins in cultured insect cells(e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell. Biol.3:2156 (1983)) and the pVL series (Lucklow and Summers Virology 170:31(1989)).

Examples of mammalian expression vectors include pWLNEO, pSV2CAT, POG44,pXT1, pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed,Nature 329:840 (19887)) and pMT2PC (Kaufman et al., EMBO J. 6:187(1987)). When used in mammalian cells, the expression vector's controlfunctions are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus and Simian Virus 40.

Viral vectors have been used in a wide variety of gene deliveryapplications in cells, as well as living animal subjects. Viral vectorsthat can be used include, but are not limited to, retrovirus,lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus,vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus,geminivirus, and caulimovirus vectors. Non-viral vectors includeplasmids, liposomes, electrically charged lipids (cytofectins), nucleicacid-protein complexes, and biopolymers. In addition to a nucleic acidof interest, a vector may also comprise one or more regulatory regions,and/or selectable markers useful in selecting, measuring, and monitoringnucleic acid transfer results (delivery to specific tissues, duration ofexpression, etc.).

In addition to the regulatory control sequences discussed above, therecombinant expression vector can contain additional nucleotidesequences. For example, the recombinant expression vector can encode aselectable marker gene to identify host cells that have incorporated thevector.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acids DNA andRNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection, DNA-loaded liposomes lipofectamine-DNAcomplexes, cell sonication, gene bombardment using high velocitymicroprojectiles, and viral-mediated transfection. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

If stable integration is desired, often only a small fraction of cells(in particular, mammalian cells) integrate the foreign DNA into theirgenome. In order to identify and select integrants, a nucleic acid thatencodes a selectable marker (e.g., resistance to antibiotics) cans beintroduced into the host cells along with the nucleic acid of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acids encodinga selectable marker can be introduced into a host cell on the samevector as that comprising the nucleic acid of interest or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection cells thathave incorporated the selectable marker gene will survive, while theother cells die).

Polypeptides and fragments of the invention can be modified for in vivouse by the addition, at the amino- and/or carboxyl-terminal ends, of ablocking agent to facilitate survival of the relevant polypeptide invivo. This can be useful in those situations in which the peptidetermini tend to be degraded by proteases prior to cellular uptake. Suchblocking agents can include, without limitation, additional related orunrelated peptide sequences that can be attached to the amino and/orcarboxyl terminal residues of the peptide to be administered. This canbe done either chemically during the synthesis of the peptide or byrecombinant technology by methods familiar to artisans of average skill.Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptides can be covalently or noncovalently coupled topharmaceutically acceptable “carrier” proteins prior to administration.

In one embodiment, the polynucleotides, vectors, polypeptides, orhomologs thereof of the invention are administered directly to asubject. Generally, the compounds of the invention will be suspended ina pharmaceutically-acceptable carrier physiological saline andadministered orally or by intravenous infusion, or administeredsubcutaneously, intramuscularly, intrathecally, intraperitoneally,intrarectally, intravaginally, intranasally, intragastrically,intratracheally, or intrapulmonarily. They can be delivered directly tothe site of the disease or disorder, such as lungs, kidney, orintestines. The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100.0 μg/kg. Widevariations in the needed dosage are to be expected in view of thevariety of polynucleotides, polypeptides, fragments, and homologsavailable and the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by injection. Variations inthese dosage levels can be adjusted using standard empirical routinesfor optimization as is well understood in the art. Administration can besingle or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-,or more fold). Encapsulation of the polynucleotides, polypeptides,fragments, and homologs in a suitable delivery vehicle (e.g., polymericmicroparticles or implantable devices) may increase the efficiency ofdelivery, particularly for oral delivery.

According to certain embodiments, the polynucleotides, vectors,polypeptides, or homologs thereof can be targeted to specific cells, oftissues in vivo. Targeting delivery vehicles, including liposomes, andviral vector systems are known in the art. For example, a liposome canbe directed to a particular target cell or tissue by using a targetingagent, such as an antibody, soluble receptor or ligand, incorporatedwith the liposome, to target a particular cell or tissue to which thetargeting molecule can bind. Targeting liposomes are described, forexample, in Ho et al., Biochemistry 25:5500 (1986); Ho et al., J. Biol.Chem. 262:13979 (1987); Ho et al., J. Biol. Chem. 262:13973 (1987); andU.S. Pat. No. 4,957,735 to Huang et al., each of which is incorporatedherein by reference in its entirety). Enveloped viral vectors can bemodified to deliver a nucleic acid molecule to a target cell bymodifying or substituting an envelope protein such that the virusinfects a specific cell type. In adenoviral vectors, the gene encodingthe attachment fibers can be modified to encode a protein domain thatbinds to a cell-specific receptor. Herpesvirus vectors naturally targetthe cells of the central and peripheral nervous system. Alternatively,the route of administration can be used to target a specific cell ortissue. For example, intracoronary administration of an adenoviralvector has been shown so be effective for the delivery of a gene cardiacmyocytes (Maurice et al., J. Clin. Invest. 104:21 (1999)). Intravenousdelivery of cholesterol-containing cationic liposomes has been shown topreferentially target pulmonary tissues (Liu et al., Nature Biotechnol.15:167 (1997)), and effectively mediate transfer and expression of genesin vivo. Other examples of successful targeted in vivo delivery ofnucleic acid molecules are known in the art. Finally, a recombinantnucleic acid molecule can be selectively (i.e., preferentially,substantially exclusively) expressed in a target cell by selecting atranscription control sequence, and preferably, a promoter, which isselectively induced in the target cell and remains substantiallyinactive in non-target cells.

Another aspect of the invention relates to a method of inhibiting sodiumabsorption through a sodium channel, comprising contacting (e.g.,binding) the sodium channel with a PLUNC protein or a functionalfragment or homolog thereof. Inhibition of sodium absorption can bemeasured by any technique known in the art or disclosed herein.

Another aspect of the invention relates to a method of increasing thevolume of fluid lining au epithelial mucosal surface, comprisingcontacting (e.g., binding) a sodium channel present on the epithelialmucosal surface with a PLUNC protein or a functional fragment or homologthereof. The volume of fluid lining an epithelial mucosal surface can bemeasured by any technique known in the art or disclosed herein.

A further aspect of the invention relates to a method of reducing thelevel of a sodium channel present on the surface of a cell, comprisingcontacting (e.g., binding) the sodium channel with a PLUNC protein or afunctional fragment or homolog thereof.

An additional aspect of the invention relates to a method of treating adisorder responsive to inhibition of sodium absorption across anepithelial mucosal surface in a subject in need thereof, comprisingdelivering to the subject a therapeutically effective amount of a PLUNCprotein or a functional fragment or homolog thereof. The disorder canbe, for example, a lung disorder (e.g., cystic fibrosis, chronicobstructive pulmonary disease, acute or chronic bronchitis, or asthma),a gastrointestinal disorder inflammatory bowel disease), a kidneydisorder, or a cardiovascular disorder.

Another aspect of the invention relates to a method of regulating saltbalance, blood volume, and/or blood pressure in a subject in needthereof, comprising delivering to the subject a therapeuticallyeffective amount of a PLUNC protein or a functional fragment or homologthereof.

III. Increasing Sodium Channel Activation

A different aspect of the invention relates to methods of preventing thebinding of a PLUNC-protein to a sodium channel, thereby allowingactivation of the sodium channel and increasing the flow of sodium ions.Thus, one aspect of the invention relates to a method of increasing theactivation of a sodium channel, comprising inhibiting the binding of aPLUNC protein to the sodium channel. In one embodiment, the sodiumchannel is an epithelial sodium channel (ENaC), e.g., human ENaC. Inanother embodiment, the sodium, channel is one that is similar insequence and/or structure to ENaC, such, as acid-sensing ion channels(ASIC). In another embodiment, inhibiting the binding of a PLUNC proteinincreases cleavage of the sodium channel by a protease, thereby leading,to activation of the channel. The binding of a PLUNC protein to thesodium channel can be measured by any method known in the art or asdisclosed herein. The activation of the sodium channel can be at leastabout 20%, e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%.

The PLUNC protein can be any protein from the PLUNC family that can bindto and inhibit the activation of sodium channel. In one embodiment, thePLUNC protein is a human PLUNC protein, as well as functional fragmentsand homologs thereof. In another embodiment, the PLUNC protein isSPLUNC1 or SPLUNC2.

The method of inhibiting the activation of a sodium channel can becarried out, e.g., on an isolated sodium channel, a sodium channel in anartificial membrane, or a sodium channel in a cell or cell line. In oneembodiment, the sodium channel is present in an isolated cell, e.g., acultured primary cell or cell line. In another embodiment, the isolatedcell is part of an epithelial cell culture, e.g., a natural orartificial epithelial lining, e.g., a cell culture in a device (such asan Ussing chamber) in which characteristics such as ion flow and/orpotential can be measured across lining or an isolated tissue or tissueculture. In a further embodiment, the cell can be present in an animal,e.g., an animal that is a disease model or a subject in need oftreatment.

In one embodiment, inhibiting the binding of a PLUNC protein comprisesdelivering a PLUNC protein inhibitor to the PLUNC protein. The PLUNCprotein inhibitor can be any compound or molecule that inhibits theability of PLUNC to bind to a sodium channel or in any other mannerinhibits the activation of the sodium channel. In one embodiment, thePLUNC protein inhibitor is an antibody that specifically recognizes thePLUNC protein, e.g., the active site of the PLUNC protein.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodycan be monoclonal or polyclonal and can be of any species, of origin,including (for example) mouse, rat, rabbit, horse, goat sheep, camel, orhuman, or can be a chimeric antibody. (See, e.g., Walker et al., Molec.Immunol. 26:403 (1989). The antibodies can be recombinant monoclonalantibodies produced according to the methods disclosed in U.S. Pat. No.4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also bechemically constructed according to the method disclosed in U.S. Pat.No. 4,676,980.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. Such fragments can beproduced by known techniques. For example, F(ab′)₂ fragments can beproduced by pepsin digestion of the antibody molecule, and Fab fragmentscan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries can be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse et al., Science 254:1275 (1989)).

Antibodies of the invention may be altered or mutated for compatibilitywith species other than the species in which the antibody was produced.For example, antibodies may be humanized or camelized. Humanized formsof non-human (e.g., murine) antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ other antigen-binding subsequences of antibodies) which containminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522 (1986); Riechmann et al.,Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol. 2:593(1992)).

Methods for humanizing, non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanammo acid residues are often referred to as “import” residues, which aretypically taken from an “import” variable domain. Humanization can beessentially performed following the method of Winter and co-workers(Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323(1988); Verhoeyen et al., Science 239:1534 (1988)), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567), wherein substantially less than anintact human variable domain has been substituted the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)).The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol. 147:86 (1991)). Similarly, human antibodies can be madeby introducing human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. Upon challenge, human antibody production isobserved, which closely resembles that seen in humans in all respects,including gene rearrangement, assembly, and antibody repertoire. Thisapproach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in thefollowing scientific publications: Marks et al., Bio/Technology 10:779(1992); Lonberg et al., Nature 368:856 (1994); Morrison, Nature 368:812(1994); Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger,Nature Biotechnical, 14:826 (1996); Lonberg and Huszar, Intern. Rev.Immunol. 13:65 (1995).

Polyclonal antibodies used to carry out the present invention can beproduced by immunizing a suitable animal (e.g., rabbit, goat, etc.) withan antigen to which a monoclonal antibody to the target binds,collecting immune serum from the animal, and separating the polyclonalantibodies from the immune serum, in accordance with known procedures.

Monoclonal antibodies used to carry out the present invention can beproduced in a hybridoma cell line according to the technique of Kohlerand Milstein, Nature 265:495 (1975). For example, a solution containingthe appropriate antigen can be injected into a mouse and, after asufficient time, the mouse sacrificed and spleen cells obtained. Thespleen cells are then immortalized by fusing them with myeloma cells orwith lymphoma cells, typically in the presence of polyethylene glycol,to produce hybridoma cells. The hybridoma cells are then grown in asuitable medium and the supernatant screened for monoclonal antibodieshaving the desired specificity. Monoclonal Fab fragments can be producedin E. coli by recombinant techniques known to those skilled in the art.See, e.g., Huse, Science 246:1275 (1989).

Antibodies specific to the target polypeptide can also be obtained byphage display techniques known in the art.

Various immunoassays can be used for screening to identity antibodieshaving the desired specificity for the polypeptides of this invention.Numerous protocols for competitive binding or immunoradiometric assaysusing either polyclonal or monoclonal antibodies with establishedspecificity are well known in the art. Such immunoassays typicallyinvolve the measurement of complex formation between an antigen and itsspecific antibody antigen/antibody complex formation). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epiptopes on the polypeptides or peptides of thisinvention can be used as well as a competitive binding assay.

Antibodies can be corrugated to a solid support (e.g., beads, plates,slides or wells formed from materials such as latex or polystyrene) inaccordance with known techniques. Antibodies can likewise be conjugatedto detectable groups such as radiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzymelabels (e.g., horseradish peroxidase, alkaline phosphatase), andfluorescence labels (e.g., fluorescein) in accordance with knowntechniques. Determination of the formation of an antibody/antigencomplex in the methods of this invention can be by detection of, forexample, precipitation, agglutination, flocculation, radioactivity,color development or change, fluorescence, luminescence, etc., as iswell known in the art.

In one embodiment, the PLUNC protein inhibitor is an aptamer thatspecifically recognizes the PLUNC protein, e.g., the active site of thePLUNC protein. Recently, small structured single-stranded RNAs, alsoknown as RNA aptamers, have emerged as viable alternatives tosmall-molecule and antibody-based therapy (Que-Gerwirth et al., GeneTher. 14:283 ((2007); Ireson et al., Mol. Cancer Ther. 5:2957 (2006)).RNA aptamers specifically bind target proteins with high affinity, arequite stable, lack immunogenicity, anti elicit biological responses.Aptamers are evolved by means of an iterative selection method calledSELEX (systematic evolution of ligands by exponential enrichment) tospecifically recognize and tightly bind their targets by means ofwell-defined complementary three-dimensional structures.

RNA aptamers represent a unique emerging class of therapeutic agents(Que-Gewirth et al., Gene Ther. 14:283 (2007); Ireson et al., Mol.Cancer Ther. 5:2957 (2006)). They are relatively short (12-30nucleotide) single-stranded RNA oligonucleotides that assume a stablethree-dimensional shape to tightly and specifically bind selectedprotein targets to elicit a biological response. In contrast toantisense oligonucleotides, RNA aptamers can effectively targetextracellular targets. Like antibodies, aptamers possess bindingaffinities in the low nanomolar to picomolar range. In addition,aptamers are heat stable, lack immunogenicity, and possess minimalinterbatch variability. Chemical modifications, such as amino or fluorosubstitutions at the 2′ position of pyrimidines, may reduce degradationby nucleases. The biodistribution and clearance of aptamers can also bealtered by chemical addition of moieties such as polyethylene glycol andcholesterol. Further, SELEX allows selection from libraries consistingof up to 10¹⁵ ligands to generate high-affinity oligonucleotide ligandsto purified biochemical targets.

In another embodiment, the PLUNC protein inhibitor is a nucleicacid-based inhibitor, e.g., a siRNA, antisense oligonucleotide,ribozyme, etc.

The term “antisense nucleolide sequence” or “antisense oligonucleotide”as used herein, refers to a nucleotide sequence that is complementary toa specified DNA or RNA sequence. Antisense oligonucleotides and nucleicacids that express the same can be made in accordance with conventionaltechniques. See, e.g., U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat. No.5,149,797 to Pederson et al. The antisense nucleotide sequence can becomplementary to the entire nucleotide sequence encoding the polypeptideor a portion thereof of at least 10, 20, 40, 50, 75, 100, 150, 200, 300,or 500 contiguous bases and will reduce the level of polypeptideproduction.

Those skilled in the will appreciate that it is not necessary that theantisense nucleotide sequence be fully complementary to the targetsequence as long as the degree of sequence similarity is sufficient forthe antisense nucleotide sequence to hybridize to its target and reduceproduction of the polypeptide. As is known in the art, a higher degreeof sequence similarity is generally required for short antisensenucleotide sequences, whereas a greater degree of mismatched bases willbe tolerated by longer antisense nucleotide sequences.

For example, hybridization of such nucleotide sequences can be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions conditions represented by a wash stringency offormamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.;conditions represented by a wash stringency of 40-45% formamide with5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and/or conditionsrepresented by a wash stringency of 50% formamide with 5×Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42° C., respectively) to the nucleotidesequences specifically disclosed herein. See e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed. (Cold Spring Harbor,N.Y., 1989).

In other embodiments, antisense nucleotide sequences of the inventionhave at least; about 70%, 80%, 90%, 95%, 97%, 98% or higher sequencesimilarity with the complement of the coding specifically disclosedherein and will reduce the level of polypeptide production.

The length of the antisense nucleotide sequence (i.e., the number ofnucleotides therein is not critical as long as it binds selectively tothe intended location and reduces transcription and/or translation ofthe target sequence, and can be determined in accordance with routineprocedures. In general, the antisense nucleotide sequence will be fromeight, ten, or twelve nucleotides in length up to about 20, 30, 50, 75or 100 nucleotides, or longer, in length.

An antisense nucleotide sequence can be constructed using chemicalsynthesis and enzymatic ligation reactions by procedures known in theart. For example, an antisense nucleotide sequence can be chemicallysynthesized using naturally occurring nucleotides of various modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleotide sequences, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleotide sequence include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thio-cytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleotide sequencecan be produced using an expression vector into which a nucleic acid hasbeen cloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest).

The antisense nucleotide sequences of the invention further includenucleotide sequences wherein at least one, or all, of theinternucleotide bridging phosphate residues are modified phosphates,such as methyl phosphonates, methyl phosphonothioates,phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Forexample, every other one of the internucleotide bridging phosphateresidues can be modified as described. In another non-limiting example,the antisense nucleotide sequence is a nucleotide sequence in which one,of all, of the nucleotides contain a 2′ lower alkyl moiety, (e.g.,C₁-C₄, linear or branched, saturated or unsaturated alkyl, such asmethyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl).For example, every other one of the nucleotides can be modified asdescribed. See also, Furdon et al., Nucleic Acids Res. 17:9193 (1989);Agrawal et al., Proc. Natl. Acad. Sci. USA 87:1401 (1990); Baker et al.,Nucleic Acids Res. 18:3537 (1990); Sprout et al., Nucleic Acids Res.17:3373 (1989); Walder and Walder, Proc. Natl. Acad. Sci. USA 85:5011(1988); incorporated by reference herein in their entireties for theirteaching of methods of making antisense molecules, including thosecontaining modified nucleotide bases).

Triple helix base-pairing methods can also be employed to inhibit PLUNCproteins. Triple helix pairing is believed to work by inhibiting theability of the double helix to open sufficiently for the binding ofpolymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (e.g., Gee et al., (1994). In: Huber et al., Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).

Small interference (si) RNA, also known as RNA interference (RNAi)molecules, provides another approach for modulating the expression ofPLUNC proteins. siRNA is a mechanism of post-transcriptional genesilencing in which double-stranded RNA (dsRNA) corresponding to a codingsequence of interest is introduced into a cell or an organism, resultingin degradation of the corresponding mRNA. The mechanism by which siRNAachieves gene silencing has been reviewed in Sharp et al., Genes Dev.15:485 (2001); and Hammond et al., Nature Rev. Gen. 2:110 (2001)). ThesiRNA effect persists for multiple cell divisions before gene expressionis regained. siRNA is therefore a powerful method for making targetedknockouts or “knockdowns” at the RNA level. siRNA has proven successfulin human cells, including human embryonic kidney and HeLa cells (see,e.g., Elbashir et al., Nature 411:494 (2001)). In one embodimentsilencing can be induced in mammalian cells by enforcing endogenousexpression of RNA hairpins (see Paddison et al., Proc. Natl. Acad. Sci.USA 99:1443 (2002)). In another embodiment, transfection of small (21-23nt) dsRNA specifically inhibits nucleic acid expression (reviewed inCaplen, Trends Biotechnol. 20:49 (2002)).

siRNA technology utilizes standard molecular biology methods. dsRNAcorresponding to all or a part of a target coding sequence to beinactivated can be produced by standard methods, by simultaneoustranscription of both strands of a template DNA (corresponding to thetarget sequence) with T7 RNA polymerase. Kits for production of dsRNAfor use siRNA are available commercially, Item New England Biolabs,Inc.; Methods of transfection of dsRNA or plasmids engineered to makedsRNA are routine in the art.

MicroRNA (miRNA), single stranded RNA molecules of about 21-23nucleotides in length, can be used in a similar fashion to siRNA tomodulate gene expression (see U.S. Pat. No. 7,217,807).

Silencing effects similar to those produced by siRNA have been reportedin mammalian ceils with transaction of a mRNA-cDNA hybrid construct (Linet al., Biochem. Biophys. Res. Commun. 281:639 (2001)), providing yetanother strategy for silencing a coding sequence of interest.

The expression of PLUNC proteins can also be inhibited using ribozymes.Ribozymes are RNA molecules that cleave nucleic acids in a site-specificfashion. Ribozymes have specific catalytic domains that possessendonuclease activity (Kim et al., Proc. Natl. Acad. Sci USA 84:8788(1987); Gerlach, et al., Nature 328:802 (1987); Forster and Symons, Cell49:211 (1987)). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Michel and Westhof, J. Mol. Biol., 216:585 (1990);Reinhold-Hurek and Sbub, Nature 357:173 (1992)). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribosyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, Nature 338:217 (1989)). For example, U.S. Pat. No. 5,354,855reports that certain isozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., Proc. Natl. Acad.Sci. USA 88:10591 (1991); Sarver et al., Science 247:1222 (1990); Sioudet al., J. Mol. Biol. 223:831 (1992)).

In another embodiment, the PLUNC protein inhibitor is a mimetic (e.g., apeptidomimetic) of the sodium channel binding site recognized by a PLUNCprotein. The term “mimetic” refers to a compound that has at least 50%of at least one biological activity of a PLUNC protein (e.g., sodiumchannel binding), at least 60%, 70%, 80%, or 90% of the biologicalactivity. The term “mimetic” as used herein is intended to beinterpreted broadly and encompasses organic and inorganic molecules.Organic compounds include, but are not limited to, small molecules,polypeptides, lipids, carbohydrates, coenzymes, aptamers, and nucleicacid molecules (e.g., gene delivery vectors, antisense oligonucleotidessiRNA, all as described above). The mimetic can further be a compoundthat is identified by any of the screening methods described below.

Peptidomimetic compounds are designed based upon the amino acidsequences of the functional polypeptide fragments. Peptidomimeticcompounds are synthetic compounds having a three-dimensionalconformation (i.e., a “peptide motif”) that is substantially the same asthe three-dimensional conformation of a selected-peptide. The peptidemotif provides the peptidomimetic compound with the ability to enhanceangiogenesis in a manner qualitatively identical to that of thefunctional fragment from which the peptidomimetic was derived.Peptidomimetic compounds can have additional characteristics thatenhance their therapeutic utility, such as increased cell permeabilityand prolonged biological half-life.

The peptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbon A,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

Another aspect of the invention relates to a method of increasing sodiumabsorption through a sodium channel, comprising inhibiting the bindingof a PLUNC protein to the sodium channel, thereby activating the sodiumchannel.

An additional aspect of the invention relates to a method of decreasingthe volume of fluid lining an epithelial mucosal surface, comprisinginhibiting the binding of a PLUNC protein to a sodium channel present inthe epithelial mucosal surface, thereby activating the sodium channel.

A further aspect of the invention relates to a method of increasing thelevel of a sodium channel present on the surface of a cell, comprisinginhibiting the binding of a PLUNC protein to the sodium channel presenton the surface of the cell.

Another aspect of the invention relates to a method of treating adisorder responsive to activation of sodium absorption in a subject inneed thereof, comprising inhibiting the activity of a PLUNC protein inthe subject. In one embodiment, the disorder is a lung disorder (e.g.,pulmonary edema), a gastrointestinal disorder, a kidney disorder, or acardiovascular disorder.

A further aspect of the invention relates to a method of regulating saltbalance, blood volume, and/or blood pressure in a subject in needthereof, comprising inhibiting the activity of a PLUNC protein in thesubject.

An additional aspect of the invention relates to a method of enhancingthe sense of taste in a subject, comprising inhibiting the activity of aPLUNC protein in the subject.

IV. Polypeptides, Polynucleotides, and Mimetics

A third aspect of the invention relates to products that can be used tocarry out the methods disclosed herein. Thus, one aspect of theinvention relates to a polypeptide consisting essentially of the sodiumchannel binding domain of a PLUNC protein. The sodium channel bindingdomain is the minimal fragment of the PLUNC protein required to havesubstantially the same binding activity to the sodium channel as thefull length PLUNC protein. The term “substantially the same bindingactivity” refers to an activity that is at least about 50% of thebinding activity of full length protein, e.g., at least about 60%, 70%,80%, or 90% of the binding activity. In one embodiment, the PLUNCprotein is a human PLUNC protein. In another embodiment, the PLUNCprotein is SPLUNC1 or SPLUNC2. In certain embodiments, the fragmentcomprises, consists essentially of, or consists of the amino acidsequence starting immediately C-terminal of the signal peptide orstarting within 1, 2, 3, 4, or 5 amino acids of the C-terminus of thesignal peptide and continuing for about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ormore amino acids or any range therein. In one embodiment, the fragmentcomprises, consists essentially of, consists of a sequence from aboutresidue 20 to about residue 41 of human SPLUNC1 (SEQ ID NO:1), e.g.,about residue 22 to about residue 39, or the corresponding sequence(e.g., the approximately 20 amino acids immediately after the signalpeptide) from another PLUNC protein. In other embodiments, the fragmentcomprises, consists essentially of, or consists of a sequence startingfrom amino acid 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 and endingwith amino acid 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 of SEQ IDNO:1. In one embodiment, the sodium channel is ENaC, e.g., human ENaC.In another embodiment, the sodium channel is one that is similar insequence and/or structure to ENaC, such as acid-sensing ion channels(ASIC).

A further aspect of the invention relates to a polynucleotide encoding apolypeptide; consisting essentially of the sodium channel binding domainof a PLUNC protein.

Another aspect of the invention relates to a vector comprising thepolynucleotide of the invention.

An additional aspect of the invention relates to a cell comprising thepolynucleotide and/or vector of the invention. In one embodiment, thecell is an isolated cell, e.g., a cultured primary cell or cell line. Inanother embodiment, the isolated cell is part of an epithelial cellculture, a natural or artificial epithelial lining, e.g., a cell culturein a device (such as an Ussing chamber) in which characteristics such asion flow and/or potential can be measured across the lining, an isolatedtissue, of tissue culture. In a further embodiment the cell can bepresent in an animal, an animal that is a disease model or a subject inneed of treatment.

A further aspect of the invention relates to a compound that mimics thesodium channel binding domain of a PLUNC protein and binds to a sodiumchannel, wherein cleavage of the sodium channel by a protease isinhibited when bound to the compound. In one embodiment, the compound isa peptidomimetic. The term “compound” as used herein is intended to beinterpreted broadly and encompasses organic and inorganic molecules.Organic compounds include, but are not limited to, small molecules,polypeptides, lipids, carbohydrates, coenzymes, aptamers, and nucleicacid molecules, (e.g., gene delivery vectors, antisenseoligonucleotides, siRNA, all as described above). The compound canfurther be a compound that is identified by any of the screening methodsdescribed below.

One aspect of the invention relates to methods of detecting disordersresponsive to modulation of sodium absorption in a subject, comprisingobtaining a sample from the subject and determining the expressionand/or activity of one or more PLUNC proteins in the sample, wherein anincrease or decrease in expression and/or activity relative to the levelof expression and/or activity in a control sample is indicative of adisorder responsive to modulation of sodium absorption and that can betreated by modulation of PLUNC proteins. In one embodiment, the sampleis from a diseased tissue such as lung, kidney or intestinal tissue. Inanother embodiment, the tissue is not diseased tissue.

In this aspect, the expression and/or activity of more than one PLUNCprotein may be determined, e.g., 2, 3, 4, or more proteins. In oneembodiment, said one or more proteins is selected from the groupconsisting of SPLUNC1 and SPLUNC2. The tissue sample may be obtained byany method known in the art, such as surgery, biopsy, lavage,aspiration, etc. The sample may be a bodily fluid, e.g., blood, serum,plasma, saliva, urine, cerebrospinal fluid, perspiration, etc. Thecontrol sample may be from a normal (i.e., non-diseased) portion of thesame tissue or cell type in the subject, from a different tissue of celltype in the subject, from a matched individual, or may be a standardderived from the average of measurements taken from a population ofsubjects.

In one embodiment, determining the expression and/or activity of one ormore PLUNC proteins comprises determining the level of a nucleic acidencoding said one or more proteins. Determining the level of a nucleicacid can be carried out by any means known in the art and as describedherein, such as Northern blots, dot blots, PCR, RT-PCR, quantitativePCR, sequence analysis, gene microarray analysis, in situ hybridization,and detection of a reporter gene.

In another embodiment, determining the expression and/or activity of oneor more PLUNC proteins comprises determining the level of said one ormore proteins. Determining the level of a protein can be carried out byany means known, in the art and as described herein, such as Westernblots, immunoblots, immunoprecipitation, immunohistochemistry,immunofluorescence, enzyme-linked immunosorbant assays, andradioimmunoassays.

In a further embodiment, determining the expression and/or activity ofone or more PLUNC proteins comprises determining the activity of saidone or more polypeptides. The activity may fee any activity associatedwith the protein, including, without limitation, sodium channel binding,activity, inhibition of sodium channel activation, and ability todecrease the number of sodium channels on the surface of a cell.

VI. Screening Assays and Animals Models

The compounds of the present invention can optionally be delivered inconjunction with other therapeutic agents. The additional therapeuticagents can be delivered concurrently with the compounds of theinvention. As used herein, the word “concurrently” means sufficientlyclose in time to produce a combined effect (that is, concurrently can besimultaneously, or it can be two or more events occurring within a shorttime period before or after each other).

Another aspect of the invention relates to a polypeptide consistingessentially of a PLUNC protein binding domain of a sodium channel. Asused herein, a PLUNC protein binding domain of a sodium channel is theminimal portion of the sodium channel amino acid sequence necessary forbinding to a PLUNC protein. In one embodiment, the PLUNC protein bindingdomain is the extracellular portion of the sodium channel.

A further aspect of the invention relates to a polynucleotide encoding apolypeptide consisting essentially of the PLUNC protein binding domainof a sodium channel.

Another aspect of the invention relates to a vector comprising thepolynucleotide of the invention.

An additional aspect of the invention relates to a cell comprising thepolynucleotide and/or vector of the invention. In one embodiment, thecell is an isolated cell, e.g., a cultured primary cell or cell line. Inanother embodiment, the isolated cell is part of an epithelial cellculture, e.g., a natural or artificial epithelial lining, e.g., a cellculture in a device (such as an Ussing chamber) in which characteristicssuch as ion flow and/or potential can be measured across the lining. Ina further embodiment, the cell can be present in an animal, e.g., ananimal that is a disease model or a subject in need of treatment.

A further aspect of the invention relates to a compound that mimics thePLUNC protein binding domain of a sodium channel and binds to a PLUNCprotein, wherein binding of PLUNC protein to the sodium channel isinhibited when bound to the compound. In one embodiment, the compound isa peptidomimetic.

Another aspect of the invention relates to a kit comprising thepolypeptide, polynucleotide, vector, cell, peptidomimetic, or compoundof invention and useful for carrying out the methods of the invention.The kit may further comprise additional reagents for carrying out themethods (e.g., buffers, containers) as well as instructions.

V. Diagnosis and Monitoring of Disorders Responsive to Modulation ofSodium Absorption

The identification of the interaction between PLUNC proteins and sodiumchannels provides targets to be used for detection and diagnosis ofdisorders responsive to modulation of sodium absorption.

The identification of binding and regulatory interactions between PLUNCproteins and sodium channels provides targets that can be used to screenfor agents that modulate binding and sodium absorption as well as modelsfor studying the process of sodium channel regulation and fluidregulation in vitro or in animals.

One aspect of the invention relates to methods of identifying a compoundthat inhibits binding of PLUNC proteins to sodium channels or mimicsbinding of PLUNC proteins to sodium channels, comprising determining thebinding of PLUNC proteins to sodium channels and/or activity of sodiumchannels in the presence and absence of a test compound, and selecting acompound that increases or decreases the binding of PLUNC proteins tosodium channels and/or activation of sodium channels relative to thelevel in the absence of the compound.

In this aspect, the assay may be a cell-based or cell-free assay. In oneembodiment, the cell may be a primary cell, e.g., an epithelial cell. Inanother embodiment, the cell is from a cell line, e.g., an epithelialcell line. The cell may be contacted with the compound in vitro (e.g.,in a culture dish) or in an animal (e.g., a transgenic animal or ananimal model). In one embodiment, the detected increase of decrease inbinding and/or activity is statistically significant, e.g., at leastp<0.05, e.g., p<0.01, 0.005, or 0.001, In another embodiment, thedetected increase or decrease is at least about 10%, 20%, 30%, 40%, 50%,60&, 70%, 80%, 90%, 100% or more.

Any desired end-point can be detected in a screening assay, e.g.,binding to the polypeptide, gene or RNA, modulation of the activity ofthe polypeptide, modulation of sodium-regulated pathways, and/orinterference with binding by a known regulator of a polynucleotide orpolypeptide. Methods of detecting the foregoing activities are known inthe art and include the methods disclosed herein.

Any compound of interest can be screened according to the presentinvention. Suitable test compounds include organic and inorganicmolecules. Suitable organic molecules can include but are not limited tosmall molecules (compounds less than about 1000 Daltons), polypeptides(including enzymes, antibodies, and Fab′ fragments), carbohydrates,lipids, coenzymes, and nucleic acid molecules (including DNA, RNA, andchimerics and analogs thereof) and nucleotides and nucleotide analogs.

Further, the methods of the invention can be practiced to screen acompound library, e.g., a small molecule library, a combinatorialchemical compound library, a polypeptide library, a cDNA library, alibrary of antisense nucleic acids, and the like, or an arrayedcollection of compounds such as polypeptide and nucleic acid arrays.

In one representative embodiment, the invention provides methods ofscreening test compounds to identify a test compound that binds to aPLUNC protein or a sodium channel. Compounds that are identified asbinding to the protein can be subject to further screening (e.g., formodulation of sodium absorption) using the methods described herein orother suitable techniques.

Also provided are methods of screening compounds to identify those thatmodulate the activity of a PLUNC protein or sodium channel. The term“modulate” is intended to refer to compounds that enhance (e.g.,increase) or inhibit (e.g., reduce) the activity of the protein (orfunctional fragment). For example, the interaction of the polypeptide orfunctional fragment with a binding partner can be evaluated. As anotheralternative, physical methods, such as NMR, can be used to assessbiological function. Activity of the PLUNC protein or sodium channel canbe evaluated by any method known in the art, including the methodsdisclosed herein.

Compounds that are identified as modulators of activity can optionallybe further screened using the methods described herein (e.g., forbinding to the PLUNC protein or sodium channel or functional fragmentthereof: polynucleotide or RNA, and the like). The compound can directlyinteract with the polypeptide or functional fragment, polynucleotide ormRNA and thereby modulate its activity. Alternatively, the compound caninteract with any other polypeptide, nucleic acid or other molecule aslong as the interaction results in a modulation of the activity of thepolypeptide or functional fragment.

The screening assay can be a cell-based or cell-free assay. Further, thePLUNC protein or sodium channel (or functional fragment thereof) orpolynucleotide can be free in solution, affixed to a solid support,expressed on a cell surface, or located within a cell.

With respect to cell-free binding assays, test compounds can besynthesized or otherwise affixed to a solid substrate, such as plasticpins, glass slides, plastic wells, and the like. For example, the testcompounds can be immobilized utilizing conjugation of biotin andstreptavidin by techniques well known in the art. The test compounds arecontacted with the polypeptide or functional fragment thereof andwashed. Bound polypeptide can be detected using standard techniques inthe art (e.g., by radioactive or fluorescence labeling of thepolypeptide or functional fragment, by ELISA methods, and the like).

Alternatively, the target can be immobilized to a solid substrate andthe test compounds contacted with the bound polypeptide or functionalfragment thereof. Identifying those test compounds that bind to and/ormodulate the PLUNC protein or sodium channel or functional fragment canbe carried out with routine techniques. For example, the test compoundscan be immobilized utilizing conjugation of biotin and streptavidin bytechniques well known in the art. As another illustrative example,antibodies reactive with the polypeptide or functional fragment can bebound to the wells of the plate, and the polypeptide trapped in thewells by antibody conjugation. Preparations of test compounds can beincubated in the polypeptide (or functional fragment)—presenting wellsand the amount of complex trapped in the well can be quantitated.

In another representative embodiment, a fusion protein can be providedwhich comprises a domain that facilitates binding of the polypeptide toa matrix. For example, glutahione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with cell lysates (e.g., ³⁵S-labeled) and the test compound,and the mixture incubated under conditions conductive to complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel detected directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level, of PLUNC protein or sodium channel or functional fragmentthereof found in the bead fraction quantitated from the gel usingstandard electrophoretic techniques.

Another technique for compound screening provides for high throughputscreening of compounds having suitable binding affinity to thepolypeptide of interest, as described in published PCT applicationWO84/03564. In this method, a large number of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with the PLUNCprotein or sodium channel or functional fragment thereof and washed.Bound polypeptide is then detected by methods well known in the art.Purified polypeptide or a functional fragment can also be coateddirectly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

With respect to cell-based assays, any suitable cell can be used,including bacteria, yeast, insect cells (e.g., with a baculovirusexpression system), avian cells, mammalian cells, or plant cells. Inexemplary embodiments, the assay is carried out in a cell line thatnaturally expresses the polynucleotide or produces the polypeptide,e.g., epithelial cells. Further, in other embodiments, it is desirableto use nontransformed cells (e.g., primary cells) as transformation mayalter the function of the polypeptide.

The screening assay can be used to detect compounds that bind to ormodulate the activity of the native PLUNC protein or sodium channel(e.g., polypeptide that is normally produced by the cell).Alternatively, the cell can be modified to express overexpress) arecombinant polypeptide or functional fragment thereof. According tothis embodiment, the cell can be transiently or stably transformed witha polynucleotide encoding the PLUNC protein or sodium channel orfunctional fragment, and can be stably transformed, for example, bystable integration into the genome of the organism or by expression froma stably maintained episome (e.g., Epstein Barr Virus derived episomes).In another embodiment, a polynucleotide encoding a reporter molecule canbe linked to regulatory element of the polynucleotide encoding a PLUNCprotein or sodium channel and used to identity compounds that modulateexpression of the polypeptide.

In a cell-based assays the compound to be screened can interact directlywith the PLUNC protein or sodium channel or functional fragment thereof(i.e., bind to it) and modulate the activity thereof. Alternatively, thecompound can be one that modulates polypeptide activity (or the activityof a functional fragment) at the nucleic acid level. To illustrate, thecompound can modulate transcription of the gene (or transgene), modulatethe accumulation of mRNA (e.g., by affecting the rate of transcriptionand/or turnover of the mRNA, and/or modulate the rate and/or amount oftranslation of the mRNA transcript.

As a further type of cell-based binding assay, the PLUNC protein orsodium channel or functional fragment thereof can be used as a “baitprotein” in a two-hybrid or three-hybrid assay (see, U.S. Pat. No.5,283,317; Zervos et al., Cell 72:223 (1993); Madura et al., J. Biol.Chem. 268:12046 (1993); Bartel et al., Biotechniques 14:920 (1993);Iwabuchi et al., Oncogene 8:1693 (1993); and PCT publicationWO94/10300), to identify other polypeptides that bind to or interactwith the polypeptide of the invention or functional fragment thereof.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the polynucleotide that encodes the PLUNCprotein or sodium channel or functional fragment thereof is fused to anucleic acid encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, optionallyfrom a library of DNA sequences, that encodes an unidentified protein(“prey” or “sample”) is fused to a nucleic acid that codes, for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact in vivo, forming a complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter sequence (e.g., LacZ), which is operably linked to atranscriptional regulatory site responsive to the transcription factor.Expression of the reporter can be detected and cell colonies containingthe functional transcription factor can be isolated and used to obtainthe nucleic acid encoding the polypeptide that exhibited binding to thePLUNC protein or sodium channel or functional fragment.

Screening assays can also be carried out in vivo in animals. Thus, asstill a further aspect, the invention provides a transgenic non-humananimal comprising an isolated polynucleotide encoding a PLUNC protein orsodium channel or functional fragment thereof, which can be producedaccording to methods well-known in the art. The transgenic non-humananimal can be from any species, including avians and non-human mammals.According to this aspect of the invention, suitable non-human mammalsinclude mice, rats, rabbits, guinea pigs, goats, sheep, pigs, andcattle. Suitable avians include chickens, ducks, geese, quail, turkeys,and pheasants.

The polynucleotide encoding the polypeptide or functional fragment canbe stably incorporated into cells within the transgenic animaltypically, by stable integration into the genome or by stably maintainedepisomal constructs). It is not necessary that every cell contain thetransgene, and the animal can be a chimera of modified and unmodifiedcells, as long as a sufficient number of cells comprise and express thepolynucleotide encoding the polypeptide or functional fragment so thatthe animal is a useful screening tool.

Exemplary methods of using the transgenic non-human animals of theinvention for in vivo screening of compounds that modulate sodiumregulation, and/or the activity of a PLUNC protein or sodium channelcomprise administering a test compound to a transgenic non-human animal(e.g., a mammal such as a mouse) comprising an isolated polynucleotideencoding a PLUNC protein or sodium channel or functional fragmentthereof stably incorporated into the genome and detecting whether thetest compound modulates sodium regulation and/or polypeptide activity(or the activity of a functional fragment). It is known in the art howto measure these responses in vivo.

Methods of making transgenic animals are known in the art. DNA or RNAconstructs can be introduced into the germ line of art avian or mammalto make a transgenic animal. For example, one or several copies of theconstruct can be incorporated into the genome of an embryo by standardtransgenic techniques.

In an exemplary embodiment, a transgenic non-human animal is produced byintroducing a transgene into the germ line of the non-human animal.Transgenes can be introduced into embryonal target cells at variousdevelopmental stages. Different methods are used depending on the stageof development of the embryonal target cell. The specific line(s) of anyanimal used should, if possible, be selected for general good health,good embryo yields, good pronuclear visibility in the embryo, and goodreproductive fitness.

Introduction of the transgene into the embryo, can be accomplished byany of a variety of means known in the art such as microinjection,electroporation, lipofection, or a viral vector. For example, thetransgene can be introduced into a mammal by microinjection of theconstruct into the pronuclei of the fertilized mammalian egg(s) to causeone of more copies of the construct to be retained in the cells of thedeveloping mammal(s). Following introduction of the transgene constructinto the fertilized egg, the egg can be incubated in vitro for varyingamounts of time, or reimplanted into the surrogate host, or both. Onecommon method is to incubate the embryos in vitro for about 1-7 days,depending on the species, and then reimplant them into the surrogatehost.

The progeny of the transgenically manipulated embryos can be tested forthe presence of the construct by Southern blot analysis of a segment oftissue. An embryo having one or more copies of the exogenous clonedconstruct stably integrated into the genome can be used to establish apermanent transgenic animal line.

Transgenically altered animals can be assayed afterbirth for theincorporation of the construct into the genome of the offspring. Thiscan be done by hybridizing a probe corresponding to the polynucleotidesequence coding for the polypeptide or a segment thereof ontochromosomal material from the progeny. Those progeny found to contain atleast one copy of the construct in their genome are grown to maturity.

Methods of producing transgenic avians are also known in the art, see,e.g., U.S. Pat. No. 5,162,215.

In particular embodiments, to create an animal model in which theactivity or expression of a PLUNC protein or sodium channel isdecreased, it is desirable to inactivate, replace or knock-out theendogenous gene encoding the polypeptide by homologous recombinationwith a transgene using embryonic stem cells. In this context, atransgene is meant to refer to heterologous nucleic acid that uponinsertion within or adjacent to the gene results in a decrease orinactivation of gene expression or polypeptide amount or activity.

A knock-out of a gene means an alteration in the sequence of a gene thatresults in a decrease of function of the gene, preferably such that thegene expression or polypeptide amount or activity is undetectable orinsignificant. Knock-outs as used herein also include conditionalknock-outs, where alteration of the gene can occur upon, for example,exposure of the animal to a substance that promotes gene alteration(e.g., tetracycline or ecdysone), introduction of an enzyme thatpromotes recombination at a gene site (e.g., Cre in the Cre-lox system),or other method for directing the gene alteration postnatally. Knock-outanimals may be prepared using methods known to those of skill in theart. See, for example, Hogan, et al. (1986) Manipulating the MouseEmbryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

A knock-out construct is a nucleic acid sequence, such as a DNA or RNAconstruct, which, when introduced into a cell, results in suppression,(partial or complete) of expression of a polypeptide encoded byendogenous DNA in the cell. A knock-out construct as used herein mayinclude a construct containing a first fragment from the 5′ end of thegene encoding a PLUNC protein or sodium channel, a second fragment fromthe 3′ end of the gene and a DNA fragment encoding a selectable markerpositioned between the first and second fragments. It should beunderstood by the skilled artisan that any suitable 5′ and 3′ fragmentsof a gene may be used as long as the expression of the correspondinggene is partially or completely suppressed by insertion of thetransgene. Suitable selectable markers include, but are not limited to,neomycin, puromycin and hygromycin. In addition, the construct maycontain a marker, such as diphtheria toxin A or thymidine kinase, forincreasing the frequency of obtaining correctly targeted cells. Suitablevectors include, but are not limited to, pBLUESCRIPT, pBR322, and pGEM7.

Alternatively, a knock-out construct may contain RNA molecules such asantisense RNA, siRNA, and the like to decrease the expression of a geneencoding a PLUNC protein or sodium channel. Typically, for stableexpression the RNA molecule is placed under the control of a promoter.The promoter may be regulated, if deficiencies in the protein ofinterest may lead to a lethal phenotype, or the promoter may driveconstitutive expression of the RNA molecule such that the gene ofinterest is silenced under all conditions of growth. While homologousrecombination between the knock-out construct and the gene of interestmay not be necessary when using an RNA molecule to decrease geneexpressions, it may be advantageous to target the knock-out construct toa particular location in the genome of the host organism so thatunintended phenotypes are not generated by random insertion of theknock-out construct.

The knock-out construct may subsequently be incorporated into viral ornonviral vector for delivery to the host animal or may be introducedinto embryonic stem (ES) cells. ES cells are typically selected fortheir ability to integrate and become part of the germ line of adeveloping embryo so as to create germ line transmission of theknock-out construct. Thus, any ES cell line that can do so is suitablefor use herein. Suitable cell lines which may be used include, but arenot limited to, the 129J ES cell line or the J1 ES cell line, The cellsare cultured and prepared for DNA insertion using methods well-known tothe skilled artisan (e.g., see Robertson (1987) In: Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. IRLPress, Washington, D.C.; Bradley et al., Curr. Topics Develop. Biol.20:357 (1986); Hogan et al., (1986) Manipulating the Mouse Embryo: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

Insertion of the knock-out construct into the ES cells may beaccomplished using a variety of methods well-known in the art,including, for example: electroporation, microinjection, and calciumphosphate treatment. For insertion of the DNA or RNA sequence, theknock-out construct nucleic acids are added to the ES cells underappropriate conditions for the insertion method chosen. If the cells areto be electroporated, the ES cells and construct nucleic acids areexposed to an electric pulse using an electroporation machine(electroporator) and following the manufacturer's guidelines for use.After electroporation, the cells are allowed to recover under suitableincubation conditions. The cells are then screened for the presence ofthe knockout construct.

Each knock-out construct to be introduced into the cell is firsttypically linearized if the knock-out construct has been inserted into avector. Linearization is accomplished by digesting the knock-outconstruct with a suitable restriction endonuclease selected to cut onlywithin the vector sequence and not within the knock-out constructsequence.

Screening for cells which contain the knock-out construct (homologousrecombinants) may be done using a variety of methods. For example, asdescribed herein, cells can be processed as needed to render DNA in themavailable for hybridization with a nucleic acid probe designed tohybridize only to cells containing the construct. For example, cellularDNA can be probed with ³²P-labeled DNA which locates outside thetargeting fragment. This technique can be used to identify those cellswith proper integration of the knock-out construct. The DNA can beextracted front the cells using standard methods (e.g., see, Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor,N.Y., 1989)). The DNA may then be analyzed by Southern blot with a probeor probes designed to hybridize in a specific pattern to genomic DNAdigested with one or more particular restriction enzymes.

Once appropriate ES cells are identified, they are introduced into anembryo using standard methods. They can be introduced usingmicroinjection, for example. Embryos at the proper stage of developmentfor integration of the BS cell to occur are obtained, such as byperfusion of the uterus of pregnant females. For example, mouse embryosat 3-4 days development can be obtained and injected with ES cells usinga micropipet. After introduction of the ES cell into the embryo, theembryo is introduced into the uterus of a pseudopregnant female mouse.The stage of the pseudopregnancy is selected to enhance the chance ofsuccessful implantation. In mice, 2-3 days pseudopregnant females areappropriate.

Germline transmission of the knockout construct may be determined usingstandard methods. Offspring resulting from implantation of embryoscontaining the ES cells described above are screened for the presence ofthe desired alteration knock-out of the PLUNC protein). This may bedone, for example, by obtaining DNA from offspring (e.g., tail DNA) toassess for the knock-out construct, using known methods, (e.g., Southernanalysis, dot blot analysis, PCR analysis). See, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold SpringHarbor, N.Y., 1989). Offspring identified as chimeras may be crossedwith one another to produce homozygous knock-out animals.

Mice are often used as animal models because they are easy to house,relatively inexpensive, and easy to breed. However, other knock-outanimals may also be made in accordance with the present invention suchas, but not limited to, monkeys, cattle, sheep, pigs, goats, horses,dogs, cats, guinea pigs, rabbits and rats. Accordingly, appropriatevectors and prompters well-known in the art may be selected and used togenerate a transgenic animal deficient in expression of PLUNC protein orsodium channel.

In another embodiment, animal models may be created using animals thatare not transgenic.

VII. Pharmaceutical Compositions

As a further aspect, the invention provides pharmaceutical formulationsand methods of administering the same to achieve any of the therapeuticeffects (e.g., modulation of sodium absorption) discussed above. Thepharmaceutical formulation may comprise any of the reagents discussedabove in a pharmaceutically acceptable carrier, e.g., a polynucleotideencoding a PLUNC protein or sodium channel or a fragment thereof, aPLUNC protein or sodium channel or fragment thereof, an antibody againsta PLUNC protein, an antisense oligonucleotide, an siRNA molecule, aribozyme, an aptamer, a peptidomimetic, a small molecule, or any othercompound that modulates the activity of PLUNC protein or sodium channel,including compounds identified by the screening methods describedherein.

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, i.e., the material can beadministered to a subject without causing any undesirable biologicaleffects such as toxicity.

The formulations of the invention can optionally comprise medicinalagents, pharmaceutical agents, carriers, adjuvants, dispersing agents,diluents, and the like.

The compounds of the invention can be formulated for administration in apharmaceutical carrier in accordance with known techniques. See, e.g.,Remington, The Science And Practice of Pharmacy (9^(th) Ed., 1995). Inthe manufacture of a pharmaceutical formulation according to theinvention, the compound (including the physiologically acceptable saltsthereof) is typically admixed with, inter alia, acceptable carrier. Thecarrier can be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose formulation, for example, atablet, which can contain from 0.01 or 0.5% to 95% or 99% by weight ofthe compound. One or more compounds can be incorporated in theformulations of the invention, which can be prepared by any of thewell-known techniques of pharmacy.

A further aspect of the invention is a method of treating subjects invivo, comprising administering to a subject a pharmaceutical compositioncomprising a compound of the invention in a pharmaceutically acceptablecarrier, wherein the pharmaceutical composition is administered in atherapeutically effective amount. Administration of the compounds of thepresent invention to a human subject or an animal in need thereof can beby any means known in the art for administering compounds.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g.,subcutaneous, intramuscuular including skeletal muscle, cardiac muscle,diaphragm muscle and smooth muscle, intradermal, intravenous,intraperitoneal), topical (i.e., both skin and mucosal surfaces,including airway surfaces), intranasal, transdermal, intraarticular,intrathecal, and inhalation administration, administration to the liverby intraportal delivery, as well as direct organ injection (e.g., intothe liver, into the brain for delivery to the central nervous system,into the pancreas, or into a tumor of the tissue surrounding a tumor).The most suitable route in any given case will depend on the nature andseverity of the condition being treated and on the nature of theparticular compound which is being used.

For injection, the carrier will typically be a liquid, such as sterilepyrogen-free water, pyrogen-free phosphate-buffered saline solution,bacteriostatic water, or Cremopbor EL[R] (BASF, Parsippany, N.J.). Forother methods of administration, the carrier can be either solid orliquid.

For oral administration, the compound can be administered in soliddosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Compounds can beencapsulated in gelatin capsules together with inactive ingredients andpowdered carriers, such as glucose, lactose, sucrose, mannitol, starch,cellulose or cellulose derivatives, magnesium stearate, stearic acid,sodium saccharin, talcum, magnesium carbonate and the like. Examples ofadditional inactive ingredients that can be added to provide desirablecolor, taste, stability, buffering capacity, dispersion or other knowndesirable features are red iron oxide, silica gel, sodium laurylsulfate, titanium dioxide, edible white ink and the like. Similardiluents can be used to make compressed tablets. Both tablets andcapsules can be manufactured as sustained release products to providefor continuous release of medication over a period of hours. Compressedtablets can be sugar coated or film coated to mask any unpleasant tasteand protect the tablet from the atmosphere, or enteric-coated forselective disintegration in the gastrointestinal tract. Liquid dosageforms for oral administration can contain coloring and flavoring toincrease patient acceptance.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the compound in a flavored base, usually sucrose andacacia of tragacanth; and pastilles comprising the compound in an inertbase such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the compound, which preparations are preferably isotonicwith the blood of the intended recipient. These preparations can containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient. Aqueousand non-aqueous sterile suspensions can include suspending agents andthickening agents. The formulations can be presented in unit\dose ormulti-dose containers, for example sealed ampoules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, saline orwater-for-injection immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound of theinvention, in a unit dosage form in a sealed container. The compounderor salt is provided in the form of a lyophilizate which is capable ofbeing reconstituted with a suitable pharmaceutically acceptable carrierto form a liquid composition suitable for injection thereof into asubject. The unit dosage form typically comprises from about 10 mg toabout 10 grams of the compound or salt. When the compound or salt issubstantially water-insoluble, a sufficient amount of emulsifying agentwhich is pharmaceutically acceptable can be employed in sufficientquantity to emulsify the compound or salt in an aqueous carrier. Onesuch useful emulsifying agent is phosphatidyl choline.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These can be prepared by admixing thecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which can be used, include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration can be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration can also be delivered byiontophoresis (see, for example, Tyle, Pharm. Res. 3:318 (1986)) andtypically take the form of an optionally buffered aqueous solution ofthe compound. Suitable formulations comprise citrate or bis\tris buffer(pH 6) or ethanol/water and contain from 0.1 to 0.2M of the compound.

The compound can alternatively be formulated for nasal administration orotherwise administered to the lungs of a subject by any suitable means,e.g., administered by an aerosol suspension of respirable particlescomprising the compound, which the subject inhales. The respirableparticles can be liquid or solid. The term “aerosol” includes anygas-borne suspended phase, which is capable of being inhaled info thebronchioles or nasal passages. Specifically, aerosol includes agas-borne suspension of droplets, as can be produced in a metered doseinhaler or nebulizer, or in a mist sprayer. Aerosol also includes a drypowder composition suspended in air or other carrier gas, which can bedelivered by insufflation from an inhaler device, for example. SeeGanderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood(1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier System6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143(1992). Aerosols of liquid particles comprising the compound can beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer, as is known to those of skill inthe art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particlescomprising the compound can likewise be produced with any solidparticulate medicament aerosol generator, by techniques known in thepharmaceutical art.

Alternatively, one can administer the compound in a local rather thansystemic manner, for example, in a depot or sustained-releaseformulation.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound orsalt thereof is an aqueous-soluble salt, using conventional liposometechnology, the same can be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed can be of anyconventional composition and can either contain cholesterol or can becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced can be reduced insize, as through the use of standard sonication and homogenizationtechniques.

The liposomal formulations containing the compounds disclosed herein orsalts thereof, can be lyophilized to produce a lyophilizate which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

In the case of water-insoluble compounds, a pharmaceutical compositioncan be prepared containing the water-insoluble compound, such as forexample in an aqueous base emulsion. In such an instance, thecomposition will contain a sufficient amount of pharmaceuticallyacceptable emulsifying agent to emulsify the desired amount of thecompound. Particularly useful emulsifying agents include phosphatidylcholines and lecithin.

In particular embodiments, the compound is administered, to the subjectin a therapeutically effective amount, as that term is defined above,Dosages of pharmaceutically active compounds can be determined bymethods known in the art, see, e.g., Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.). The therapeutically effectivedosage of any specific compound will vary somewhat from compound tocompound, and patient to patient, and will depend upon the condition ofthe patient and be route of delivery. As a general preposition, a dosagefrom about 0.1 to about 50 mg/kg will have therapeutic efficacy, withall weights being calculated based upon the weight of the compound,including the cases where a salt is employed. Toxicity concerns at thehigher level can restrict intravenous dosages to a lower level such asup to about 10 mg/kg, with all weights being calculated based upon theweight of the compound, including the cases where a salt is employed. Adosage from about 10 mg/kg to about 50 mg/kg can be employed for oraladministration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg canbe employed for intramuscular injection. Particular dosages are about 1μmol/kg to 50 μmol/kg, and more particularly to about 22 μmol/kg and to33 μmol/kg of the compound for intravenous or oral administration,respectively.

In particular embodiments of the invention, more than one administration(e.g., two, three, four, or more administrations) can be employed over avariety of time intervals (e.g., hourly, daily, weekly, monthly, etc.)to achieve therapeutic effects.

The present invention finds use in veterinary and medical applications.Suitable subjects include both avians and mammals, with mammals beingpreferred. The term “avian” as used herein includes, but is not limitedto, chickens, ducks, geese, quail, turkeys, and pheasants. The term“mammal” as used herein includes, but is not limited to, humans,bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.Human subjects include neonates, infants, juveniles, and adults.

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLE 1 Experimental Methods

Tissue procurement and cell culture: Cells were harvested by enzymaticdigestion from human bronchial tissue as previously described under aprotocol approved by the UNC School of Medicine IRB (Tarran et al., J.Gen. Physiol. 127:591 (2006)). Human excess donor lungs and excisedrecipient lungs were obtained at the time of lung transplantation fromportions of main stem or lumbar bronchi and cells were harvested byenzymatic digestion. All preparations were maintained at an air liquidinterface in a modified bronchial epithelial medium and used 2-5 weeksafter seeding on 12 mm T-Clear inserts (Corning Costar) coated withhuman placental type VI collagen (Sigma). Phosphate buffered saline(PBS) was used for washing human bronchial epithelial culture mucosalsurfaces.

Identification of SPLUNC1: Airway surface liquid was collected bylavaging human bronchial epithelial cultures with 100 μl PBS at 37° C.for 15 min. The lavage was then centrifuged for 5 min at 4000 rpm toremove dead cells and the supernatants were incubated overnight on anend-over-end rotator at 4° C. with trypsin-agarose heads(Sigma)±aprotinin (Sigma). The beads were eluted using 30 μl Laemmlibuffer, boiled at 95° C. for 5 min, separated on a 15% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) gel per theUniversity of North Carolina-Duke Michael Hooker Proteomics Centerstandard procedures (proteomics.unc.edu/protocol.shtml). Visible bandswere excised and prepared for mass spectrometry analysis by MALDI-MS/MSas described previously (Loiselle et al., J. Proteome Res. 4:992(2005)).

Mitroelectrode studies. A single-barreled potential difference-sensingelectrode was placed in the airway surface liquid by micromanipulatorand used in conjunction with a macroelectrode in the serosal solution tomeasure transepithelial voltage using a voltmeter (World PrecisionInstruments). Trypsin (2 U/ml; Sigma) was added mucosally as a drypowder in perfluorocarbon to test for changes in regulation of ENaC aspreviously described (Tarran et al., J. Gen. Physiol. 127:591 (2006).Transepithelial resistance was routinely measured using the EVOM system(WPI) as previously described (Tarran et al., J. Gen. Physiol. 127:591(2006)).

Oocyte studies. Xenopus laevis oocytes were harvested and injected asdescribed (Donaldson, et al., J. Biol. Chem. 277:8338 (2002)).Defolliculated healthy stage V-VI oocytes were injected with 0.3 ng ofcRNA of each ENaC subunit. Injected oocytes, were kept in modifiedBarth's a saline (in mM: 88 NaCl, 1 KCl, 2.4 NaHCO₃, 0.3 Ca(NO₃)₂, 0.41CaCl₂, 0.82 MgSO₄, and 15 HEPES, adjusted to pH 7.35 with Tris). Oocyteswere studied 24 hr after injection using the two electrode voltage clamptechnique as previously described (Donaldson, et al., J. Biol. Chem.277:8338 (2002)). Oocytes were clamped at a holding potential of 60 mV.The change in amiloride-sensitive whole cell current as an indicator ofENaC activity was determined by subtracting the corresponding currentvalue measured in the presence of 10 μM amiloride from that measuredbefore the application of amiloride.

Western Blotting: Airway surface liquid collected as described above wasalso placed in protease-inhibitor cocktail (Roche) for Western blotting.The protein concentration was determined using the BCA Assay (Pierce).To obtain SPLUNC1 from Xenopus oocytes, oocytes were lysed in Laemmlibuffer or oocyte media was directly sampled and placed in Laemmlibuffer. Proteins were resolved using SDS-PAGE and transferred to aPolyvinylidene Fluoride (PVDF) membrane. The membrane was then probedusing αSplunc1 or αV5 antibodies and a Donkey anti-Mouse HRP antibody(R&D). The blots were then incubated with ECL reagents (Pierce).

Binding Assay: JME nasal epithelial cells which did not express ENaCwere stably infected with a lentivirus containing yfp-αENaC or emptyvector (control). JME cells were incubated with varying concentrationsof Texas red-labeled SPLUNC1 for 30 min followed by a 5× wash with PBS.After this time images were acquired with a Nikon Ti-S invertedmicroscope and were quantified to obtain specific and non-specific usingImage J. Data were then fitted with a Hill Plot to obtain the K_(d).

shRNA-induced knockdown of SPLUNC1: Our strategy was to select shRNAsequences from Dharmacon that targeted SPLUNC1 effectively by usingtransient siRNA in an immortalized human airway epithelial cell line(denoted AALEB) (Lundberg et al., Oncogene 21:4577 (2002)). We thengenerated viruses encoding the most effective siRNA. Passage-1 airwaycells surviving one week of selection were then trypsinized and plateddown on 12 mm T-clear inserts and differentiated under air liquidinterface conditions. At the time of the functional assays. We measuredairway surface liquid SPLUNC1 protein levels by Western blot to verifystable knockdown. An anti-luciferase shRNA-expressing adenovirus wasinfected separately as a control.

Confocal microscopy: To label airway surface liquid, Ringer containingTexas Red-dextran (2 mg/ml; Invitrogen) was added to human bronchialepithelial culture mucosal surfaces. Perfluorocarbon was added mucosallyto prevent evaporation of the airway surface liquid and the cultureplaced in a chamber containing 100 μl Ringer on the stage of a Leica SP5confocal microscope with a 63× glycerol immersion objective. 5 pointsper culture were scanned and average airway surface liquid heightdetermined. For confocal microscopy human bronchial epithelial cultureswere bathed serosally in a modified Ringer solution containing (mM): 116NaCl, 10 NaHCO₃, 5.1 KCl, 1.2 CaCl₂, 1.2 MgCl₂, 20 TES, 10 glucose, pH7.4). At all other times, human bronchial epithelial cultures weremaintained in a modified BEGM growth medium which contained 24 mM NaHCO₃gassed with CO₂. Perfluorocarbon (FC-77) was obtained from 3M and had noeffect on ASL height as previously reported.

Flp-in HEK293 cell culture and SPLUNC1 protein purification: Flp-InHEK293 cells (Invitrogen) were transfected withpcDNA5/FRT/V5-his-TOPO/hSplunc1 vector. SPLUNC1-expressing clones wereselected using hygromycin, isolated, and analyzed for expression. Theclones which stably express SPLUNC1 were cultured in T75 flasks in DMEMHmedia containing 5% Fetal Bovine Serum and at 37° C. in 5% CO₂.His-tagged SPLUNC1 was purified from cultured media by dialyzing themedia into the His-Select Binding Buffer (50 mM sodium phosphate, pH8.0, 300 mM sodium chloride, 10 mM imidazole) overnight at 4° C.,incubating the dialyzed media with His-Select Nickel Affinity Matrix(Sigma): for 4 hours at 4° C. on an end-over-end rotator in the presenceof protease inhibitors (Roche), applied to a column, and washed with 40ml of His-Select Binding Buffer. SPLUNC1 was then eluted from the Cobaltaffinity matrix in 0.5 ml fractions with 600 mM imidazole in His-SelectBinding Buffer. Purified SPLUNC1 was then exchanged into Ringer.Cultured media from FlpIn HEK293 cells lacking SPLUNC1 was processed inthe same way as media from FlpIn HEK293-SPLUNC1 cells and used ascontrol for experiments where purified SPLUNC1 was added.

Fluorogenic Assays: To determine whether SPLUNC1 inhibited trypsinactivity we assayed cleavage of the D-tert-butyldicarbonate-Gln-Ala-Arg-7-methoxycoumarin-4-yl)acetyl (BGAR-MCA)fluorogenic substrate in Ringer (Peptides Int.) excited at 350 nm andemission collected at 460 nm in a 96 well plate reader format (Wallac1420 VICTOR2). For cell-free assays, reactions were carried out in 50 μlRinger in a 96 well, plate format with 100 μM BGAR-MCA. To measureendogenous protease activity in human bronchial epithelial cultures, 30μl Ringer with 100 μM BGAR-MCA were placed directly onto the mucosalsurfaces of human bronchial epithelial cultures grown on 12 mm T-clearinserts and the cultures were assayed in 12 well plates.

Co-Immunoprecipitation: Xenopus oocytes were injected with eitherHA-N-Terminus or V5-C-Terminus (HA-NT/V5-CT) tagged subunits incombination with wild type (WT) untagged rat αβγENaC subunits (0.3 ngcRNA each) with or without V5-tagged SPLUNC1 and CAP1 (1 ng cRNA each).After 24 h, 40 eggs per experimental condition were lysed with buffercontaining (in mM): 20 Tris, 50 NaCl, 50 NaF, 10 β-glycerophosphate, 5Na4P₂O₇ pyrophosphate, 1 EDTA, pH 7.5 and protease inhibitors (complete,Roche), aprotinin (Sigma). Cell lysates were prepared by passing theeggs through a 27G1/2needle twice and by centrifugation at 3,600 rpm for10 minutes at 4° C. Supernatants were transferred to new tubes andsamples were spun at 14,000 rpm for 20 minutes at 4° C. Supernatantswere discarded and pellets were solubilized in (mM) 50 Tris, 100 NaCl,0.1% Triton X-100, 0.1% NP-40, 20 NaF, 10 Na₄P₂O₇, 10 EDTA+proteaseinhibitor cocktail (Sigma), pH 7.5. Total inputs were taken from wholecell samples representing 4% of total protein. Solubilized proteins wereincubated with 50 μl of protein A and 5 μl of anti-HA antibody (Covance)overnight while tumbling at 4° C. Samples were washed, three times with(mM) 150 NaCl 50 Tris pH 7.5 buffer. Laemmli buffer was added andsamples were loaded on a 15% gradient Tris-glycine gel after incubationfor 10 minutes at 96° C. Samples were transferred to PVDF membranes andWestern blot analysis was performed using an anti-V5 (Invitrogen)monoclonal antibody. SPLUNC1 bound to ENaC only when ENaC and SPLUNC1lysates were used. Uninjected and SPLUNC1 lysates lacking ENaC were bothnegative for co-immunoprecipitation.

Generation of yfp-αENaC expressing cell line and SPLUNC1 binding assay.The yfp-αENaC construct has previously been shown to function normally(Berdiev et al., J. Biol. Chem. 282:36481 (2007)) and was subcloned intoa lentiviral vector (pQCXIN). JME nasal epithelial cells expressfunctional ENaCs (Tong et al., Am. J. Physiol. Lung Cell. Mol. Physiol.287:L928 (2004)). However, this attribute is lost after severalpassages. Thus, passaged JME cells that no longer expressed ENaC werestably infected with a lentivirus containing yfp-αENaC or an emptyvector as a control and the presence or absence of αENaC was confirmedusing an antibody that was constructed “in-house” that was directedagainst αENaC (FIG. 8).

Recombinant SPLUNC1 was labeled with Texas red according to themanufacturer's instructions (Pierce) and was freshly labeled on the dayof each binding experiment. JME cells were plated on 12 mm T-Clearculture inserts (Corning Costar) and were cultured until confluent.Cultures were then washed 3× with PBS to remove cellular debris andincubated with varying concentrations of Texas red-SPLUNC1 for 30 min inPBS²⁺ (with Ca²⁺ and Mg²⁺; 10 μl total volume) followed by a 5× washwith PBS. After this time yfp (514 nm excitation) and Texas redfluorescence (590 nm excitation) were imaged under a 60× water objectiveon a Nikon Ti-S inverted microscope equipped with an Orea CCD camera(Hamamatsu) switchable filter wheels (Ludl). Background fluorescence wassubtracted from all images and the mean thresholded intensity wasquantified to obtain specific and non-specific binding using Image J.

PCR and primer sequences: PCR was performed using Amplitaq GoldMastermix (ABI) and primers specific for SPLUNC1 at a finalconcentration of 200 nM. The primers used were: forward5′-ctgatggccaccgtcctat-3′ (SEQ ID NO:3) and reverse5′-aggtggatcctctcctgctt-3′ (SEQ ID NO:4). The reaction was performedaccording to the manufacturer's instructions with an extension time of30 seconds for an Eppendorf MasterCycler. Water was used as a negativecontrol, and SPLUNC1 cDNA as a positive control. Human cDNA was preparedfrom 200 ng of RNA using, superscript II (Invitrogen), and 1 μl was usedfor each reaction. A product of the appropriate size ˜150 bp wasdetected by gel electrophoresis.

Statistical analyses: All data are presented as the mean±SE for aexperiments. Airway cultures derived from three or more separate donorswere used for each study and each oocyte study was repeated an threeseparate occasions. Differences between means were tested forstatistical significance using paired or an paired t tests or their nonparametric equivalent as appropriate to the experiments. From suchcomparisons, differences yielding P≦0.05 were judged to be significant.All binding assays were fitted to the Hill equation.

EXAMPLE 2 Identification of SPLUNC1

Based on the ability of normal human bronchial epithelial cultures toregulate airway surface liquid height to 7 μm, which was paralleled by adecrease in trypsin-sensitive ENaC activity, we speculated that asoluble protease inhibitor is present so the airway surface liquidduring normal airway surface liquid volume homeostasis. We searched forpotential protease inhibitors/ENaC regulators by incubatingtrypsin-coated beads with airway surface liquid and performing aproteomic analysis. Airway surface liquid was collected by lavaginghuman bronchial epithelial cultures with 100 μl PBS at 37° C. for 15min, incubated overnight at 4° C. with trypsin-agarose beads±aprotinin,separated on a 15% SDS page gel and visualized with a silver stain (FIG.1). Bands were removed from the gel for analysis by mass spectrometryand the identities of these proteins are listed in Table 2. Of note,SPLUNC1 was visible as a ˜26 kD protein (band 1) and as a ˜19 kDfragment (band 2) and its binding to trypsin was moderately out-competedby the addition of the protease inhibitor aprotinin (FIG. 1). The massspectrometry analysis allowed us to identify SPLUNC1 as one of the majorproteins that bound to the trypsin-beads (FIG. 2A; Table 2). Thepresence of SPLUNC1 was confirmed in airway surface liquid by Westernblot (FIG. 2B).

TABLE 2 Band No. Protein Name Accession No. 1 SPLUNC1 AAF70860 2 SPLUNC1AAF70860 AY513239 AAR89906 3 Complement C3 Precursor C3HU 4 HypotheticalProtein Q8WVW5_HUMAN

To better study SPLUNC1, we stably transfected V5/6His-tagged SPLUNC1into HEK293 cells and purified secreted V5/6His-SPLUNC1 from HEK293media over a nickel column. Recombinant SPLUNC1 (rSPLUNC1) could bedetected using the anti-V5 antibody (FIG. 3) and a brief (30 min)incubation with trypsin resulted in the appearance of cleavage productsof C-terminally V5-tagged rSPLUNC1, indicating that SPLUNC1 is asubstrate for serine proteases (FIG. 3). To test whether SPLUNC1 wascapable of altering airway ion transport, we then measured thetransepithelial voltage under thin film conditions in human bronchialepithelial cultures±rSPLUNC1 with time. Washing the mucosal surface ofhuman bronchial epithelial cultures with PBS has previously been shownto maximally activate ENaC (Tarran et al., J. Gen. Physiol. 127:591(2006)) and also removes endogenous SPLUNC1 (FIG. 3B). Under theseconditions, 20 μl of Ringer containing 50 ng/ml of rSPLUNC1significantly reduced the transepithelial voltage (FIG. 2C). Incontrast, a purified SPLUNC1-free fraction of HEK293 media was withouteffect (FIG. 2C). To confirm that this inhibition was due to alteredENaC regulation, we exposed human bronchial epithelial cultures totrypsin for 30 min after the 1 hr rSPLUNC1 exposure. Trypsin was withouteffect in the control human bronchial epithelial cultures, suggestingthat ENaC remained fully active. However, mucosal trypsin exposuresignificantly raised the transepithelial voltage in the SPLUNC1-exposedgroup, suggesting that ENaC had been inhibited by rSPLUNC1 (FIG. 2C).Inhibition of the transepithelial voltage occurred at identical ratesfollowing both rSPLUNC1 and aprotinin addition and the effects of thesecompounds were not additive. However, in both cases, these effects werereversed by trypsin-exposure (FIG. 2D, 2E). Taken together, these datasuggest (i) that both molecules operated through a common pathway and(ii) that this was an ENaC-specific effect (FIG. 2E, 3E). Further, whenairway surface liquid was left to accumulate on human bronchialepithelial culture surfaces for 24 h (i.e., the cultures were notpre-washed with Ringer), the transepithelial voltage was significantlylower than in washed cultures and rSPLUNC1 addition was without furthereffect, suggesting that the spontaneous accumulation of an endogenousinhibitor in the airway surface liquid reduces the transepithelialvoltage and that maximum inhibition is reached at steady state (FIG.2E).

Since SPLUNC1 binds to trypsin (FIG. 3), we explored the possibilitythat SPLUNC1 is proteolytically cleaved in the process. There was both a˜1 kDa and a 10 kDa shift in mobility of C-terminal V5-tagged rSPLUNC1.The ˜16 kDa band, likely corresponds to the 2^(nd) SPLUNC1 band detectedby mass spectrometry (FIG. 1 and Table 2), suggesting that endogenousSPLUNC1 is cleaved in airway surface liquid.

EXAMPLE 3 SPLUNC1 Regulates Ion Transport

To test whether passive fluxes were affected by SPLUNC1 exposure, wemeasured the effects of amiloride on transepithelial voltage vs.transepithelial electrical resistance±rSPLUNC1. Amiloride reduced thetransepithelial voltage by 54% (n=12) and rSPLUNC1 addition to the sameamiloride-treated cultures was without further effect (n=12). Inparallel both amiloride and rSPLUNC1 increased the transepithelialelectrical resistance by ˜33% and again the effects were not additive,suggesting that amiloride and SPLUNC1 both act on ENaC in the apicalmembrane and increase the apical membrane resistance in human bronchialepithelia, rather than by affecting paracellular transport (FIG. 4).

To further investigate how SPLUNC1 regulated ion transport and ENaC inparticular, we expressed αβγENaC in Xenopus laevis oocytes and eitherexposed oocytes to rSPLUNC1 or co-injected SPLUNC1 cRNA into the oocyteswith αβγENaC. ENaC currents were reduced by when oocytes were incubatedwith rSPLUNC1 prior to recording (FIG. 5A). Due to the larger volumesrequired for oocyte incubations, SPLUNC1 was added at a 10 a lowerconcentration than in the human bronchial epithelial cultures (5 ng/ml).Similarly, co-expression of αβγENaC and SPLUNC1 also resulted in ENaCinhibition by ˜70% (FIG. 5A). SPLUNC1 could not be detected in mediafrom oocytes injected with αβγENaC (FIG. 5B). However, SPLUNC1 wasreadily detected in the media after coinjection of SPLUNC1 and αβγENaCcRNAs (FIG. 5B). Since SPLUNC1 could be detected in the oocyte media(FIG. 5), it is likely that co-expressed SPLUNC1 was secreted by theoocytes and inhibited ENaC externally in a similar fashion to rSPLUNC1.

To test whether SPLUNC1 specifically inhibited ENaC, we either exposedCFTR-expressing oocytes to 5 ng/ml rSPLUNC1 or co-expressed SPLUNC1 andCFTR. In both cases, we co-expressed CFTR with the β2 adrenergicreceptor (β2AR) which can be stimulated with isoproterenol to raise cAMPand stimulate CFTR (Uezono et al., Receptors Channels 1:233 (1993)).Following 10 μM isoproterenol exposure, CFTR was robustly activated andunlike with ENaC, rSPLUNC1 exposure or injection of SPLUNC1 cRNA had noinhibitory effect on CFTR activity, suggesting that the inhibitoryeffects of SPLUNC1 are specific for ENaC (FIG. 5C).

EXAMPLE 4 SPLUNC1 Inhibits Cleavage of ENaC

Since SPLUNC1 bound to trypsin-agarose beads (FIG. 1) and affected thetrypsin sensitivity of ENaC (FIG. 2C), we next tested whether SPLUNC1could alter serine protease activity. Despite rSPLUNC1 being capable ofinhibiting ENaC by ˜70% in both human bronchial epithelia and oocytes(FIGS. 1 and 2), 50 ng/ml rSPLUNC1 had only a modest affect (˜10%) onthe ability of either 1.0 or 0.3 U/ml trypsin to cleave: a fluorogenicsubstrate (Di-tert-butyldicarbonate-Gln-Ala-Arg-7-methoxycoumarin-4-yl)acetyl; BGAR-MCA), unlike2 U/ml aprotinin which inhibited trypsin activity by ˜100% (FIG. 6A).Airway epithelia express serine proteases and mucosal addition of Ringersolution containing BGAR-MCA to human bronchial epithelial culturesresulted in spontaneous BGAR-MCA cleavage with time that was inhibitedby aprotinin addition, confirming that serine proteases are indeedactive on the mucosal surface of airway epithelia (FIG. 6B). rSPLUNC1had no significant affect on BGAR-MCA cleavage in human bronchialmucosal surfaces, suggesting that SPLUNC1 does not inhibit ENaC byinhibiting serine protease activity (FIG. 6B).

Proteolytic cleavage of α and γ subunits is required for ENaC activation(Mueller et al., J. Biol. Chem. 282:33475 (2007); Adebamiro et al., J.Gen. Physiol. 130:611 (2007)) and since CAP2 is highly expressed inhuman bronchial epithelial cultures (Tarran et al., J. Gen. Physiol.127:591 (2006)), we tested whether SPLUNC1 could alter α and γ ENaCcleavage by CAP2. Due to the relative scarcity of purified rSPLUNC1, weelected to co-express SPLUNC1 and ENaC rather than use rSPLUNC1 forsubsequent oocyte studies since SPLUNC1 is secreted at sufficientquantities from oocytes to inhibit ENaC (FIG. 5). When αβγENaC and CAP2were co-expressed in oocytes, both full-length ENaC subunits and cleavedα and γ fragments were detected with a V5 antibody (FIG. 7A, 7B).However, in the presence of SPLUNC1, only full-length α and γ ENaCsubunits could be observed, suggesting that SPLUNC1 protects ENaC fromproteolytic cleavage despite SPLUNC1 having no observable intrinsicanti-protease activity (FIG. 7A, 7B). Since we probed with a V5antibody, SPLUNC, which is also V5-tagged was visible as a 26 kD band.However, SPLUNC1 could be differentiated from ENaC cleavage fragmentsbased on its size and position on the gel (FIG. 7A, 7B). To confirm thatSPLUNC1 prevented functional activation of ENaC by CAPS, we co-expressedENaC±SPLUNC1 with prostasin (CAP1) and CAP2 both of which are present inthe airways. As previously described, both CAPs significantly increasedbasal ENaC currents (Vuagniaux et al., J. Gen. Physiol. 120:191 (2002)).However, the ability of both proteases to activate ENaC wassignificantly reduced by SPLUNC1 (FIG. 7C). Similarly, trypsin-exposurealso increased ENaC activity and this simulation was attenuated bySPLUNC1 (FIG. 2D). The furin-insensitive α_(R203,231K,)β,γ_(R138K) ENaCmutant was also inhibited by SPLUNC1, suggesting that this effect is notmediated by convertases such as furin (Hughey et al., J. Biol. Chem.279:18111 (279:18111 (2004)) (FIG. 7E).

EXAMPLE 5 SPLUNC1 Binds to ENaC

Since SPLUNC1 prevented cleavage and activation of ENaC, but did notappear to be a serine protease inhibitor in the same fashion asaprotinin, we hypothesized that SPLUNC1 could specifically bind to ENaCto protect it from proteolysis. To test this hypothesis, we utilized anairway cell line (JME cells) that had been extensively passaged and didnot express ENaC, which we used to measure non-specific binding afterinfection with a lentivirus containing an empty vector. To measurespecific binding, we then infected these cells with a lentiviruscontaining yfp-tagged αENaC since the αENaC subunit alone has previouslybeen shown to form functional Na⁺ channels, albeit with a significantlysmaller conductance (Kizer et al., Proc. Natl. Acad. Sci USA 94:1013(1997); McDonald et al., Am. J. Physiol. 268:C1157 (1995)). Stable αENaCexpression was confirmed by western blot (FIG. 8). To qualitatively testthe relationship between yfp-αENaC expression and SPLUNC1 binding, weplated JME cells on glass coverslips and incubated these cells withvarying concentrations of Texas red-labeled rSPLUNC1. As can be seen inFIG. 9A/Texas red-rSPLUNC1 and yfp-αENaC clearly colocalize whilstrSPLUNC1 binding to JME cells infected with the lentiviral vector alonewas reduced and more diffuse (FIG. 9A). To quantitatively address thisissue, we then polarized these cells on filters for seven days. We thenincubated these polarized cells with varying concentrations of Texasred-rSPLUNC1 for 30 min followed by a 5× wash with PBS. The subsequentbinding isotherm shows a clear difference between non-specific (emptyvector-transfected) binding which did not saturate and specific(vfp-αENaC) binding which was significantly greater and saturable (FIG.9B). Using this graph, we calculated that the K_(d) was 55 ng/ml (FIG.9B). Thus, if SPLUNC1 is indeed a volume sensor in the airways, thisdata suggests that it will be able to change ENaC activity over a narrowrange of concentrations.

Xenopus oocytes are autofluorescent, making this type of fluorescentbinding assay difficult. To test whether SPLULNC1 could also bind toENaC subunits in Xenopus oocytes, we co-expressed HA-tagged N-terminusand V5-tagged C-terminus (HA-NT/V5-CT) αβγ ENaC subunits in combinationwith wild type (WT) untagged subunits and V5-tagged SPLUNC1 (forexample, αHA-NT/V5-CT,β,γ-ENaC±SPLUNC1-V5) and immunoprecipitated ENaCusing anti-HA monoclonal antibodies. We then probed for V5-taggedSPLULNC1 and found that SPLUNC1 bound to all three ENaC subunits (FIG.10). Thus, rather than being a protease inhibitor, we propose thatSPLUNC1 protects α and γ ENaC form being cleaved by serine proteases,perhaps being cleaved itself in the process.

We have previously shown that human bronchial epithelial culturesrapidly absorb excess airway surface liquid and then absorption slowsand a steady state airway surface liquid height of ˜7 μm is maintained(Tarran et al, (J. Biol. Chem. 280:35751 (2005)). To ask if endogenousSPLUNC1 was required as part of this homeostatic mechanism, we knockeddown SPLUNC1 using two different air anti-SPLUNC1 shRNA sequences thatwere incorporated into retroviruses that were used to infect humanbronchial epithelial cultures. The shRNAs had the following sequences.

shRNA No. 1 Sense (SEQ ID NO: 5) AUAAAGUCCUGCCUGAGUUUUshRNA No. 1 Anti-sense (SEQ ID NO: 6) 5′PAACUCAGGCAGGACUUUAUUUshRNA No. 2 Sense (SEQ ID NO: 7) GCAGGAAGCUUGACAAAUGUUshRNA No. 2 Anti-sense (SEQ ID NO: 8) 5′PCAUUUGUCAAGCUUCCUGCUU

Successful knockdown was confirmed by qPCR and western blot (FIGS. 5A,B) and since no difference in knockdown was detected between eachsequence, the subsequent results were pooled. Human bronchial epithelialcultures infected with a control shRNA (anti-luciferase), rapidlyabsorbed a test solution of 20 μl Ringer until an airway surface liquidheight of 7 μm was reached, after which time absorption slowed andairway surface liquid height was maintained at 7 μm as has previouslybeen described for non-infected human bronchial epithelial cultures(FIG. 11C, 11D) (Tarran et al., J. Gen. Physiol. 127:591 (2006); Tarranet al., (J. Biol. Chem. 280:35751 (2005)). This regulation wasparalleled by a decline in the transepithelial voltage which could berestored by mucosal exposure to trypsin (FIG. 11E). Importantly,cultures lacking SPLUNC1 failed to regulate airway surface liquid heightwith time and exhibited increased airway surface liquid absorptionduring the initial phase followed by failure to maintain steady-stateairway surface liquid height at 7 μm (FIG. 11C, 11D). Further, thetransepithelial voltage failed to decline in human bronchial epithelialcultures lacking SPLUNC1 and remained both elevated andtrypsin-insensitive, suggesting that ENaC remained fully activated.Regulation of both airway surface liquid height and the transepithelialvoltage was restored by the addition of 50 ng/ml rSPLUNC1 to the airwaysurface liquid (FIG. 11C-11E) suggesting that SPLUNC1 indeed acts asreporter molecule in the airway surface liquid that regulates ENaCactivity to maintain appropriate airway surface liquid volume control.

We propose that SPLUNC1 binds specifically to an extracellular domain ofENaC, preventing the channel from being cleaved and activated by serineproteases. If has been proposed that the extracellular loops of ENaCplay a role in channel gating and the α and γ subunits of ENaC have beenreported to contain short inhibitory segments that are removed duringproteolytic cleavage to activate the channel (Carattino et al, J. Biol.Chem. 283:25290 (2008); Carattino et al., Am. Physiol. Renal Physiol.294:F47 (2008)). We speculate that ENaC subunits that have already beencleaved by extracellular serine proteases are likely to beSPLUNC1-insensitive. However, as new ENaCs are inserted in the plasmamembrane, SPLUNC1 binds to them, preventing their cleavage and resultingin a decline in ENaC-mediated currents. The onset of inhibition (30-60min; FIG. 2C, 2D) is comparable with aprotinin-inhibition rates in humanbronchial epithelial cultures (Bridges et al., Am. J. Physiol. Lung CellMol. Physiol. 281:L16 (2001); Donaldson et al., J. Biol. Chem. 277:8338(2002)) and is consistent with this model. While the cleavage model isgenerally accepted, it is also conceivable that proteins which bind tothe extracellular loops of ENaC could induce sufficient conformationalchanges to modulate the activity of the channel. While the slow kineticsof the inhibition with SPLUNC1 are most compatible with the cleavagehypothesis (FIG. 2), we cannot as yet formally exclude the possibilitythat SPLUNC1 can bind to ENaC and either induce a conformational changein the extracellular loops and/or the pore to block the channel ordirectly block the channel pore itself. Further, in this study, we didnot differentiate between possible effects of SPLUNC1 on the number ofENaC channels their open probability. Since both basal andprotease-activated ENaC currents were reduced in the presence ofSPLUNC1, the relative, increase from basal to protease-activatedcurrents is similar±SPLUNC1 (FIG. 7). Thus, we cannot exclude thepossibility that SPLUNC1 decreases the number of ENaC channels in theplasma membrane. If this was the ease, then the pool of surface ENaCsavailable to be cleaved would be reduced, which could explain both thereduction in ENaC cleavage and protease-activated currents in thepresence of SPLUNC1.

In addition to being expressed in the airways, ENaC is also expressed inaldosterone-sensitive epithelial cells in the colon and kidney where itplays an important role in the control of sodium balance, blood volume,and blood pressure (Kunzelmann et al., Physiol. Rev. 82:245 (2002);Rossier et al., Annu. Rev. Physiol. 64:877 (2002)). In the colon, theprimary flux is in the absorptive direction. However, ion transport canswitch from being absorptive to being secretory to help regulate saltbalance (Charney et al., Am. J. Physiol. 247:G1 (1984); Garty et al.,Physiol. Rev. 77:359 (1997)). In the kidney, ENaC is the rate-limitingstep for salt reabsorption in the collecting duct (20) and aldosteroneinduces a shift in the molecular weight of γENaC from 85 kDa to ≈75 kDa,consistent with physiological proteolytic clipping of the extracellularloop (Masilamani et al., J. Clin. Invest. 104:R19 (1999)). As theiracronym suggests, palate lung and nasal epithelial clone (PLUNC) familymembers expression was thought to be limited to a few specific tissues(Bingle et al., Biochim. Biophys. Acta 1493:363 (2000)). We performedPGR to determine whether SPLUNC1 was also expressed in otherENaC-expressing tissues. Interestingly, SPLUNC1 was highly expressed inthe kidney and colon, but was not expressed in the stomach (FIG. 12)suggesting that SPLUNC1 is expressed in other ENaC-expressing tissuesbeyond the lung/palate and nasal epithelia. Thus, SPLUNC1 expression inthese tissues could potentially add an additional layer of regulation tofurther modulate ENaC activity and salt absorption.

SPLUNC1 expression is increased in cystic fibrosis lungs, especially inthe surface epithelium of the proximal and distal airways (Bingle etal., Respir. Res. 8:79 (2007)) and this upregulation may be due to theincreased inflammation in seen CF lungs (Chmiel et al., Respir. Res. 4:8(2003)). However, cystic fibrosis lungs are typified by Na⁺hyperabsorption and mucus dehydration (Boucher, Pflugers Arch. 445:495(2003)) so it is unlikely that SPLUNC1 exerts any significant inhibitoryeffect on ENaC under these conditions. Further, we have previouslydemonstrated that cystic fibrosis bronchial epithelial cultures do notdecrease ENaC activity with time (Tarran et al., J. Gen. Physiol.127:591 (2006); Tarran et al., (J. Biol. Chem. 280:35751 (2005)). CFTRexpression is not required for SPLUNC1 to inhibit ENaC, as demonstratedin our oocytes studies, suggesting that this inability of SPLUNC1 toregulate ENaC is not an innate property of cystic fibrosis airways (FIG.5A). However, the serine proteases that activate ENaC are unregulated incystic fibrosis airway epithelia (Myerburg et al., Am. J. Physiol. LungCell Mol. Physiol. 294:L932 (2008)) and neutrophil elastase, which alsoactivates ENaC, is increased in cystic fibrosis airways (Birrer et al.,Am. J. Respir. Crit. Cure Med. 150:207 (1994); Caldwell et al., Am. J.Physiol. Lung Cell Mol. Physiol. 288:L813 (2005); Knostan et al., Am. J.Respir. Crit. Care Med. 150:448 (1994)). Thus, it is possible that theexcessive protease upregulation seen in cystic fibrosis airways(Myerburg et al., Am. J. Physiol. Lung Cell Mol. Physiol. 294:L932(2008)) interferes with the normal regulation of ENaC by SPLUNC1 andother potential ENaC regulators and thereby shifts the balance fromanti-proteases and less ENaC activity to a protease-replete state withmore ENaC activity, overwhelming the ability of SPLUNC1 to inactivateENaC and contributing to cystic fibrosis airway surface liquid volumedepletion.

In summary, we have identified SPLUNC1 as a novel extracellular proteininhibitor of ENaC that is present in the airway surface liquid. Innormal airways, SPLUNC1 is highly expressed in submucosal glands withmoderate expression in surface epithelium of the proximal airways withlittle expression in the distal airways (Bingle et al., Respir. Res.8:79 (2007)). Thus, we propose that SPLUNC1 is secreted glands andsurface epithelium where it serves as a reporter molecule whose dilutionor concentration can adjust ENaC activity to regulate airways hydrationand mucus clearance. Since SPLUNC1 is secreted by proximal airways, wepropose that this regulation primarily occurs in the proximal airways,with little effect in the distal airways.

EXAMPLE 6 SPLUNC2 Inhibits ENaC

SPLUNC1 or SPLUNC2 were expressed in Xenopus oocytes as described inExample 3. The effect of SPLUNC1, SPLUNC2, and LPLUNC1 onamiloride-sensitive currents was measured (FIG. 13). Current isdisplayed relative to amiloride-sensitive current from α,β,γENaC-expressing oocytes (CTRL, white bar, n=18). Oocytes co-expressingSPLUNC1 showed a ˜70% reduction in ENaC current (p<0.0001; n=22). Thoseco-expressing SPLUNC2 (n=22) exhibited borderline significance (i.e.,not significant with ANOVA and significant (p=0.045) with an unpairedt-test). Oocytes co-expressing LPLUNC1 (n=17) had no significant ENaCcurrent reduction in this system. The * denotes p<0.05 differencecompared to control oocytes expressing α,β,γ ENaC.

EXAMPLE 7 Reduction Inhibits SPLUNC1 Activity

The effect of reducing agents on SPLUNC1 activity was tested in primaryhuman bronchial epithelial cultures (FIG. 14). Cultures were prewashedto remove endogenous SPLUNC1 and the basal PD was measured (control,ctrl), then, either 50 ng/ml recombinant SPLUNC1 or recombinant SPLUNC1that had been reduced with DTT was added, and the PD was remeasured onthe same cultures 45 min later. Pretreatment with DTT abolished SPLUNC1activity. Western blot analysis under non-denaturing conditions showingthat reduced (i.e., DTT-treated) SPLUNC1 migrates along the gel at adifferent rate than non-denatured SPLUNC1 (FIG. 15).

EXAMPLE 8 SPLUNC1 May Decrease the Number of ENaC Channels

The effect of SPLUNC1 on the number of ENaC channels in the plasmamembrane was tested by expressing SPLUNC1 and αENaC Xenopus oocytes.After surface biotinylation of αENaC, total lysate was prepared andlysate from 3-4 eggs was separated on a 10% gel. FIG. 16A shows thatplasma membrane ENaC is decreased following coexpression with SPLUNC1 inoocytes. The addition of MTSET to ENaC containing the βS518C mutantincreases ENaC P_(o) to 1.0 when coexpressed in oocytes yet the overallcurrent is still reduced by SPLUNC1 expression, suggesting that ENaC hasbeen internalized (FIG. 16B).

EXAMPLE 9 Identification of the SPLUNC1 Active Site

SPLUNC1 is a 256 amino acid protein that contains an N-terminal signalsequence that enables the protein to be secreted extracellularly.C-terminal truncation mutants of SPLUNC1 were prepared and examined forthe ability to inhibit ENaC channels in Xenopus oocytes. The mutantproteins are shown in FIG. 17A. The N-terminal signal peptide sequence(amino acids 1-19) and putative active site (amino acids 22-39) areindicated. Each mutant was tested in the oocyte current inhibition assaydescribed in Example 3. The activity of the truncation mutants is shownin FIG. 17B. Significant inhibition (all p<0.0001) of ENaC was observedwith the full-length, 60%, 30%, and 15% proteins. However, deletion of89% or 98% of SPLUNC1 prevented its inhibition of ENaC. As amino acids1-19 of SPLUNC1 are a signal sequence that enables the protein to besecreted, this leaves a predicted inhibitory peptide of about 29 aminoacids (residues 20-41) as the likely active site for SPLUNC1. Notably,all truncates were secreted into the extracellular media, as checked bywestern blot, consistent with the hypothesis that SPLUNC1 actsextracellularly.

It is interesting to note that amino acids 22-30 of SPLUNC1 share ˜40%homology with the inhibitory fragments of ENaC that are excised uponproteolytic cleavage and are known to inhibit ENaC (FIG. 17A). However,since SPLUNC1 acts reducing the number of ENaC channels at the plasmamembrane, whilst the α26 and γ43 subunits of ENaC act by reducing theopen probability of ENaC, their mechanism of action appears to markedlydiffer.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method of inhibiting the activation of a sodium channel, comprisingcontacting, a sodium channel with a PLUNC protein or a functionalfragment thereof.
 2. The method of claim 1, wherein the sodium channelis an epithelial sodium channel (ENaC).
 3. (canceled)
 4. The method ofclaim 1, wherein the PLUNC protein is SPLUNC1 or SPLUNC2. 5-8.(canceled)
 9. The method of claim 1, wherein contacting the sodiumchannel with a PLUNC protein comprises delivering the PLUNC protein, ora functional fragment thereof to a cell comprising the sodium channel.10. The method of claim 1, wherein contacting the sodium channel with aPLUNC protein comprises delivering a polynucleotide encoding the PLUNCprotein or a functional fragment thereof to a cell comprising the sodiumchannel. 11-15. (canceled)
 16. A method of increasing the volume offluid lining an epithelial mucosal surface, comprising contacting asodium channel present on the epithelial mucosal surface with a PLUNCprotein or a functional fragment thereof.
 17. The method of claim 16,wherein the sodium channel is ENaC. 18-19. (canceled)
 20. A method oftreating a disorder responsive to inhibition of sodium absorption acrossan epithelial mucosal surface in a subject in need thereof, comprisingdelivering to the subject a therapeutically effective amount of a PLUNCprotein or a functional fragment thereof.
 21. The method of claim 20,wherein the sodium channel is ENaC. 22-86. (canceled)